Lysosomal Phospholipase A2 (LPLA2) Activity as a Diagnostic and Therapeutic Target for Identifying and treating Systemic Lupus Erythematosis

ABSTRACT

The present invention is directed to methods for diagnosis and treatment of systemic lupus erythematosus and drug-induced systemic lupus erythematosus. More specifically, the specification describes methods using a lysosomal phospholipase A2 in methods for the diagnosis and treatment of autoimmune disorders such as systemic lupus erythematosus and drug-induced systemic lupus erythematosus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of 12/495,209, filed Jun. 30, 2009,which claims the benefit under 35 U.S.C. 119(3) of U.S. ProvisionalPatent Application No. 61/076,913, filed Jun. 30, 2008.

STATEMENT REGARDING FEDERAL SPONSOR RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.RO1DK055823 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF INVENTION

The present invention is generally directed to methods for diagnosingand treating systemic lupus erythematosus (SLE) and symptoms thereof.

BACKGROUND OF THE RELATED ART

Systemic lupus erythematosus (SLE) is an autoimmune disease that affectsmultiple organs. The clinical manifestations are variable, but are ofteninvolve the skin, kidneys, liver, lungs, heart, and lymphoid organs.LPLA2 knockout mice (Hiraoka et al., Mol Cell Biol 26, 6139-6148, 2006)display a highly robust late onset lymphoproliferation that phenocopiesSLE. More specifically, the mouse phenotype is characterized bysplenomegaly, lymphadenopathy, penumonitis, hepatitis, andglomerulonephritis with renal insufficiency and immunoglobulin, C3 andClq depositions. These mice are also characterized by high levels ofcirculating immunoglobulins and high titers of anti-nuclear antibodies(ANA) and anti-double stranded DNA antibodies (anti-dsDNA). Thisstriking phenotype raises the question of whether LPLA2 mediates theimmunological processes that are aberrantly regulated in lupus.

Therefore, there remains a need in the art for a better understanding ofthe causes of SLE and for the identification of new therapeuticinterventions for such autoimmune disorders.

SUMMARY OF THE INVENTION

Lysosomal phospholipase A2 (LPLA2) has several unique features,including lipase and transacylase activities, lysosomal localization,and acidic pH optimum. Based on these features, double conditional genetargeting was employed to elucidate the function of LPLA2. LPLA2deficient mice were generated by the systemic deletion of exon 5 of theLPLA2 gene, which encodes the lipase motif essential for LPLA2 activity.LPLA2 null mice displayed features of systemic lupus erythematosus (SLE)that included late onset lymphoproliferative phenotype, expansion oflymphoid tissues, renal failure and glomerulonephritis, high circulatingimmunoglobulin levels, positive antinuclear antibodies (ANA) andanti-double stranded DNA antibodies, and the accumulation of undigestedapoptotic or tingible bodies in macrophages.

The present invention unexpectedly demonstrates that LPLA2 mediates animmunological process that is aberrantly regulated in SLE. Thus, thepresent invention demonstrates a role for loss of LPLA2 enzyme activityas a marker of active SLE. Work described herein demonstrates thatdecreased levels of LPLA2 can be used as a diagnostic and therapeutictarget for both identifying and treating symptoms of SLE.

As used herein, “LPLA2” means full-length lysosomal phospholipase A2enzymes, also referred to as Group XV phospholipase A2, regardless ofspecies of origin, as well as all fragments, derivatives andenzymatically active variants thereof. The term “variant” refers toadditions, deletions or substitutions of amino acids. The term“derivative” encompasses additional chemical modification of LPLAs, suchas chemical modification of amino acids or incorporation of modifiedamino acids, conjugation to hydrophilic polymers, or conjugation toother chemical moieties.

“LPLA2 enzymatic activity” refers to the ability of LPLA2 to degradeextracellular phospholipids to form free fatty acids andlyso-phophatidylcholine and lyso-phosphatidylethanolamine.

“LPLA2 catalytic consensus sequence” refers to amino acids -A-X-S-X-G-at positions 196-200 of SEQ ID NO: 1.

“LPLA2 autoantibodies” refers to antibodies produced by an individualthat are immunospecific to the individual's own LPLA2 protein.

“Tingible body macrophages” refers to enlarged macrophages resultingfrom the inability of spleen cells to clear apoptotic cells.

As used herein, “SLE” includes systemic lupus erythematosus or any otherautoimmune disease displaying the symptom of accumulation of tingiblebody macrophages. “Drug-induced lupus” refers to the onset of lupussymptoms such as the accumulation of tingible body macrophages uponchronic treatment with certain drugs.

The present invention provides methods for reducing the accumulation oftingible body macrophages in an individual comprising the step ofcontacting a cell having an accumulation of tingible body macrophageswith an agent having LPLA2 enzymatic activity in an amount and over atime to reduce the accumulation of tingible body macrophages.

In one aspect, the agent having LPLA2 activity is a protein having anamino acid sequence selected from SEQ ID NOs: 1-288.

In another aspect, the agent is a mammalian LPLA2 enzyme. In oneembodiment, the agent is a mammalian LPLA2 enzyme having an amino acidsequence selected from SEQ ID NOs: 1-288 or an enzymatically activefragment, derivative or variant thereof.

In yet another aspect, the agent is a human LPLA2 enzyme. In oneembodiment, the agent is a human LPLA2 enzyme having the amino acidsequence in SEQ ID NO: 1 or an enzymatically active fragment, derivativeor variant thereof.

In one embodiment, the agent is a LPLA2 enzyme comprising one or moremannose residues or one or more mannose-6-phosphate residues.

In another embodiment, the agent is a human LPLA2 enzyme or variant orderivative thereof comprising the catalytic site corresponding to aminoacids 196-200 of SEQ ID NO: 1 (-A-X-S-X-G-).

In some embodiments, the human LPLA2 enzyme, variant or derivativethereof further comprises a cysteine bond corresponding to the cysteinebond between the Cys at position 65 and the Cys 89 of SEQ ID NO: 1.

In one aspect, the agent is a human LPLA2 enzyme or variant orderivative thereof comprising an amino acid sequence having at least65%, 70%, 75%, 80%, 85%, 90%, 95% or higher identity to the amino acidsequence of a fragment of SEQ ID NO: 1 that is at least 50, 75, 100,125, 150, 175, 200 or 225 residues in length and that comprises thecatalytic site corresponding to amino acids 196-200 of SEQ ID NO: 1(-A-X-S-X-G-).

In another aspect, the agent is a LPLA2 enzyme comprising the catalyticconsensus sequence -A-X-S-X-G-. In still another aspect, the agent is aLPLA2 enzyme comprising the catalytic consensus sequence correspondingto amino acids 196-200 (-A-X-S-X-G-) of SEQ ID NO: 1. In relatedaspects, the LPLA2 enzyme comprises a cysteine bond corresponding to thecysteine bond between the Cys at position 65 and the Cys 89 of SEQ IDNO: 1.

The present invention further provides methods for treating a conditionassociated with aberrant LPLA2 activity in an individual comprising thestep of administering an agent having LPLA2 enzymatic activity to saidindividual in an amount effective to alleviate the condition.

In one aspect, the invention provides methods for treating a patientdiagnosed with a disorder characterized by the intracellularaccumulation of tingible body macrophages comprising administering tothe patient an effective amount of an agent having LPLA2 enzymaticactivity.

In another aspect, the condition is aberrant LPLA2 enzymatic activity.In another aspect, the condition is aberrant LPLA2 mRNA transcription.In still another aspect, the condition is the accumulation of tingiblebody macrophages. In yet another aspect, the condition is SLE orsystemic lupus erythematosus. In another aspect, the condition isdrug-induced lupus.

In one embodiment, the individual has a polymorphism in the LPLA2 genecorresponding to SEQ ID NO: 1 that reduces expression or activity ofLPLA2. The human genomic DNA sequence encoding human LPLA2 enzyme is setforth in SEQ ID NO: 289. The human genomic DNA sequence is 15715 bases.The human mRNA sequence is predicted to correspond to nucleotides 1 . .. 210, 3947 . . . 4103, 9576 . . . 9694, 9939 . . . 10037, 10423 . . .10647, and 13803 . . . 15715). The coding region is predicted tocorrespond to nucleotides 84 . . . 210, 3947 . . . 4103, 9576 . . .9694, 9939 . . . 10037, 10423 . . . 10647, and 13803 . . . 14314. Thehuman cDNA sequence encoding human LPLA2 enzyme is set forth in SEQ IDNO: 290. In some embodiments, the individual has a polymorphism at aposition in the gene that encodes the catalytic site corresponding toamino acids 196-200 of SEQ ID NO: 1. In other embodiments, theindividual has a polymorphism that results in truncation of the LPLA2enzyme or loss of cationic amino acids. In yet another embodiment, theindividual has a polymorphism in a regulatory control region associatedwith the LPLA2 gene that results in loss of LPLA2 enzyme expression.

In a further aspect, the agent having LPLA2 activity is a protein havingan amino acid sequence selected from SEQ ID NOs: 1-288.

In another aspect, the agent is a mammalian LPLA2 enzyme. In oneembodiment, the agent is a mammalian LPLA2 enzyme having an amino acidsequence selected from SEQ ID NOs: 1-288 or an enzymatically activefragment, derivative or variant thereof.

In yet another aspect, the agent is a human LPLA2 enzyme. In oneembodiment, the agent is a human LPLA2 enzyme having the amino acidsequence in SEQ ID NO: 1 or an enzymatically active fragment, derivativeor variant thereof.

In one embodiment, the agent is a human LPLA2 enzyme, variant orderivative thereof, comprising the catalytic site corresponding to aminoacids 196-200 (A-X-S-X-G) of SEQ ID NO: 1. In another aspect, the agentis a LPLA2 enzyme, variant or derivative thereof comprising thecatalytic consensus sequence -A-X-S-X-G-.

In some embodiments, the LPLA2 enzyme, variant or derivative furthercomprises a cysteine bond corresponding to the cysteine bond between theCys at position 65 and the Cys 89 of SEQ ID NO: 1.

In one aspect, the agent is a human LPLA2 enzyme, variant or derivativethereof comprising an amino acid sequence having at least 65%, 70%, 75%,80%, 85%, 90%, 95% or higher identity to the amino acid sequence of afragment of SEQ ID NO: 1 that is at least 50, 75, 100, 125, 150, 175,200 or 225 residues in length and that comprises the catalytic sitecorresponding to amino acids 196-200 of SEQ ID NO: 1 (-A-X-S-X-G-). Inrelated aspects, the LPLA2 enzyme further comprises a cysteine bondcorresponding to the cysteine bond between the Cys at position 65 andthe Cys 89 of SEQ ID NO: 1

In one embodiment, the individual has a polymorphism that reducesexpression or activity of LPLA2. In another embodiment, the individualhas a polymorphism at a position corresponding to amino acids 196-200 ofSEQ ID NO: 1. In a further embodiment, the individual has a polymorphismthat results in truncation of the LPLA2 enzyme or loss of cationic aminoacids. In yet another embodiment, the individual has a polymorphism inthe regulatory control region of LPLA2 that results in loss of LPLA2enzyme expression.

In yet another aspect, the agent is an LPLA2 enzyme comprising one ormore mannose residues.

The present invention also provides methods for screening potentialtherapeutics that decrease intracellular levels of tingible bodymacrophages comprising the step of measuring the level of intracellulartingible body macrophages in the LPLA2 null mouse model in the presenceor absence of a test compound where a decrease in the level ofintracellular tingible body macrophages in the presence of the testcompound compared to the level of intracellular tingible bodymacrophages in the absence of the test compound identifies the testcompound as a potential therapeutic.

The present invention also provides methods for diagnosing SLE in anindividual comprising the step of determining LPLA2 enzymatic activityin a sample from an individual, wherein a LPLA2 enzymatic activity thatis decreased in the individual compared to a LPLA2 enzymatic activity ina normal individual is suggestive of SLE and wherein said normalindividual is known not to suffer from SLE. As used here throughout,“normal individual” refers to an individual known not to suffer from aspecified disease.

The present invention further provides methods for diagnosing SLE in anindividual comprising the step of detecting LPLA2 autoantibodies in asample from an individual, wherein a detecting increased LPLA2autoantibodies in the individual compared to detecting LPLA2autoantibodies in a normal individual is suggestive of SLE and whereinsaid normal individual is known not to suffer from SLE.

The present invention provides methods for diagnosing SLE in anindividual comprising the step of detecting LPLA2 enzymatic activity inthe individual, wherein a LPLA2 enzymatic activity that is decreased inthe individual compared to prior LPLA2 enzymatic activity in the sameindividual is suggestive of SLE.

The present invention also provides methods for diagnosing SLE in anindividual comprising the step of detecting LPLA2 autoantibodies in theindividual, wherein detecting increased LPLA2 autoantibodies in theindividual compared to prior detection of LPLA2 autoantibodies in thesame individual is suggestive of SLE.

The present invention provides methods for determining susceptibility toSLE in an individual comprising the step of determining LPLA2 enzymaticactivity in a sample from the individual, wherein a decreased LPLA2enzymatic activity in the individual compared to LPLA2 enzymaticactivity in a normal individual indicates susceptibility to SLE.

The present invention further provides methods for determiningsusceptibility to SLE in an individual comprising the step of detectingLPLA2 autoantibodies in a sample from the individual, wherein detectingincreased LPLA2 autoantibodies in the individual compared to detectingLPLA2 autoantibodies in a normal individual indicates susceptibility toSLE.

The present invention also provides methods for determiningsusceptibility to SLE in an individual comprising the step of detectingLPLA2 enzymatic activity in a sample from the individual, wherein aLPLA2 enzymatic activity that is decreased in the individual compared toprior LPLA2 enzymatic activity in the same individual is suggestive ofsusceptibility to SLE.

The present invention further provides methods for determiningsusceptibility to SLE in an individual comprising the step of detectingLPLA2 autoantibodies in a sample from the individual, wherein detectingincreased LPLA2 autoantibodies in the individual compared to priordetection of LPLA2 autoantibodies in the same individual is suggestiveof susceptibility to SLE.

Also provided by the present invention are methods for determining theprogression of SLE in an individual comprising the step of determiningLPLA2 enzymatic activity in samples from the individual taken over time,wherein a decrease in LPLA2 enzymatic activity in samples taken overtime in the individual is suggestive of SLE progression.

The present invention also provides methods for determining theprogression of SLE in an individual comprising the step of detectingLPLA2 autoantibodies in samples from the individual taken over time,wherein an increase in LPLA2 autoantibodies in the samples taken overtime in the individual is suggestive of SLE progression.

In various embodiments of methods of the invention, LPLA2 mRNA levelsand/or LPLA2 protein levels are used to measure LPLA2 enzymatic activityin the sample.

In various aspects of the methods, the sample is a bodily fluid, tissueand/or organ of said individual.

In other aspects of the methods, the tissue is a spleen specimen.

In another aspect of the methods, the fluid is serum or plasma.

The foregoing summary is not intended to define every aspect of theinvention, and additional embodiments are described in other sections,such as the Detailed Description. The entire document is intended to berelated as a unified disclosure, and it should be understood that allpossible combinations of features described herein may be contemplated,even if the combination of features are not found together in the samesentence, or paragraph, or section of this document.

Moreover, the invention includes any one or all embodiments of theinvention that are narrower in scope in any way than the variationsdefined by specific paragraphs herein. For example, where certainaspects of the invention are described as a genus, it should beunderstood that every member of a genus is, individually, an embodimentof the invention, and that combinations of two or more members of thegenus are embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Pathways for lysosomal phospholipase A2. In the presence of anacceptor such as ceramide (N-acetylsphingosine (NAS)) the enzyme behavesas a transacylaselphosphlipase A2, akin to LCAT. With only water as anacceptor, LPLA2 behaves as a traditional phospholipase A2.

FIG. 1B. Comparison of the catalytic triads and disulfide linkages seenin LPLA2 and LCAT.

FIG. 1C. Comparison of deduced amino acid sequences of human, mouse, andbovine LPLA2s. Position 1 refers to the first amino acid residue of thepredicted coding , region in human and mouse. The dashed boxes indicatethe N-glycosylation site consensus motifs, and the solid box indicates alipase motif. The shaded boxes indicate the amino acids Ser, Asp, and His composing the catalytic triad. The axial line is a putative signalpeptide cleavage site. Asterisks denote identical amino acids amongthese three species.

FIG. 2. Gross anatomical changes in 20 month old LPLA2−/− knockout andLPLA2+/+ wild type mice.

FIG. 3. Histological changes in 20 month old LPLA2−/− knockout andLPLA2+/+ wild type mice. Tissues sections were stained with periodicacid Schiff reagent. Pink staining cells represent lipid ladenmacrophages.

FIG. 4A. Immunofluorescent staining of LPLA2−/− knockout and LPLA2+/+wild type mouse kidneys for Ig heavy chain and Clq.

FIG. 4B. Anti-dsDNA titers for 3-4 month old, 12-14 month old and 24month old LPLA2+/+ wild type and LPLA2 null mice.

FIG. 4C. ANA staining of HeLa cells from 12 month old LPLA2+/+ wild typeand LPLA2−/− knockout mice. Both homogenous and speckled nuclearstainings are observed in assays using the knockout mouse sera.

FIG. 4D. Identification of tingible bodies associated with macrophagesin spleens from LPLA2−/− knockout mice. Frozen sections were stained forCD68 (red) and apoptotic bodes with a TUNEL stain (green).

FIG. 5A-5C. Immunofluorescence staining of macrophages from LPLA2 nullmice, treated with recombinant LPLA2. LPLA2 was added to the null cellsfor 2 hours. The macrophages were then fixed and stained with eitheranti-LPLA2 monoclonal antibody (green) (FIG. 5A) or an antibody againstthe lysosomal marker LAMP-1 (red) (FIG. 5B). Alex-flour coupledsecondary antibodies were used to assess uptake and co-localization(FIG. 5C).

FIG. 6. 24 hour protein excretion in knockout (KO) LPLA2−/− mice versuswild type (WT) LPLA2+/+ mice.

FIGS. 7A-7H. The binding, endocytosis, and clearance of apoptoticthymocytes by peritoneal macrophages from wild type (WT) mice (FIGS.7A-7D) and knockout (KO) mice (FIGS. 7E-7H). Time dependent changes inendocytosed thymocytes were detected by anti-CD68 (red) and TUNEL assay(green).

FIG. 8. Phagocytic index of macrophages from WT and KO mice, as measuredby the number of apoptotic bodies/macrophage. The graphed valuesrepresent the mean+/−SD (n=8) per time point. * denotes p<0.05.

FIGS. 9A-9D. B and T cell proliferation studies in 6 or 8 month old WTand KO mice.

FIGS. 10A-10B. Effects of the cationic amphiphilic drugs PDMP andamiodarone on 1-O-acylceramide synthase activity (FIG. 10A) and cellularphospholipids content (FIG. 10B).

FIGS. 11A-11B. Effect of negatively charged lipid on LPLA2 activity.Liposomes consisting of. Di-octanoyl-PC (DOPC)/N-acetylsphingosine (NAS)or DOPC/sulfatide/NAS were incubated with LPLA2 enzyme, and the reactionproduct, 1-O-oleoyl-NAS was plotted against time (FIG. 11A). Liposomesconsisting of DOPC/galactosylceramide/NAS or DOPC/sulfatide/NAS withdifferent molar ratios of sulfatide were incubated with LPLA2 enzyme,and the reaction product, 1-O-oleoyl-NAS was plotted against molarratio.

FIG. 12A. Effect of ion strength on LPLA2 activity. 1-O-oleoyl-NASplotted against NaCl concentration (mM).

FIG. 12B. Effect of pH on LPLA2 activity. 1-O-oleoyl-NAS plotted againstpH.

FIG. 12C. Effect of cationic amphiphilic drug on LPLA2 activity.Liposomes consisting of DOPC sulfatide and NAS were pre-incubated withdifferent concentrations of a cationic amphiphilic drug, amiodarone andthen incubated with LPLA2 enzyme. 1-O-oleoyl-NAS plotted againstconcentration of drug (μM).

FIG. 13A-13C. Co-sedimentation of LPLA2 with liposomes. Liposomesconsisting of DOPC or DOPC/sulfatide were incubated with varying amountsof LPLA2 and the reaction product was separated by SDS-PAGE (FIG. 13A).Liposomes consisting of DOPC, DOPC/sulfatide or glactosylceramide(GalCer) were incubated with LPLA2 and the reaction product was analyzedby SDS-PAGE (FIG. 13B). Liposomes consisting of DOPC or DOPC/sulfatidewere incubated with LPLA2 at varying pH with or without NaCl oramiodarone (AMIOD) and the reaction product was analyzed by SDS-PAGE.

DETAILED DESCRIPTION OF INVENTION

Systemic lupus erythematosus remains a poorly understood autoimmunedisorder. Much progress has been made in the last twenty years inidentifying genetic models that resemble the lupus phenotype. However,this previous work has not readily been translated into new diagnostictools for better defining lupus patients or into new therapeutics forthe treatment of these unfortunate patients. The identification hereinof a secreted, lysosomal protein where the loss of function plays animportant role in SLE has several important implications. First, as asecreted protein, serum LPLA2 activity may be a valuable marker ofdisease activity. Second, the existence of autoantibodies to LPLA2 maybe mechanistically important in a subset of patients with SLE. Third, ifa deficiency, inherited or acquired, in LPLA2 underlies the developmentof SLE in a subset of patients, then the protein itself may be a usefultherapeutic agent. Mannose-terminated lysosomal proteins are the basisfor enzyme replacement strategies for a growing list of lysosomalstorage diseases. These include type I Gaucher disease, Fabry diseaseand Hunter syndrome. Previous studies demonstrating the avid binding,incorporation, and trafficking of LPLA2 in macrophages suggest that thisstrategy might be applied to SLE as well.

The present invention is directed to methods of treating disorderscharacterized by the intracellular accumulation of tingible bodymacrophages by administering compositions comprising LPLA2, includingfragments, variants or derivatives thereof, compositions that augment,increase or otherwise stimulate the activity of LPLA2, and compositionsthat increase or otherwise stimulate the expression of LPLA2. Suchcompositions may be used for catabolizing phospholipids and/orincreasing digestion or clearance of apoptotic bodies in mammaliantissue and/or reducing accumulation of tangible body macrophages inmammalian tissue. It is therefore contemplated that such compositionswill be useful for ameliorating signs and symptoms of a disorder thatinvolves the accumulation of tingible body macrophages, or reducingcomplications experienced by patients suffering from the disorder.Disorders characterized by intracellular accumulation of tangible bodymacrophages include systemic lupus erythematosis, drug-induced lupus,neonatal lupus, and cutaneous lupus, rheumatoid arthritis,

In a related aspect, the invention provides methods for identifyingadditional agents that decrease intracellular levels of tingible bodymacrophages through augmenting, increasing or otherwise stimulating theactivity of LPLA2, or increasing or otherwise stimulating the expressionof LPLA2.

In yet another related aspect, the invention provides methods fordiagnosis of, or the determination of the susceptibility to, SLE orsystemic lupus erythematosus by assaying body fluid or tissue samplesfrom patients for the presence of LPLA2 autoantibodies or for reducedLPLA2 expression or activity. Similar methods are provided formonitoring progression of SLE or systemic lupus erythematosus.

The present application demonstrates the involvement of a particularlysosomal phospholipase A2 in the digestion and clearance of apoptoticbodies. Mice lacking LPLA2 exhibit glomerulonephritis with proteinuria,renal failure and associated with immune complex deposition thatincludes C3, Clq, and Ig,. Macrophages in the spleen of LPLA2 null micedisplay very high levels of TUNEL positive, tingible bodies representingendocytosed but undegraded apoptotic bodies. Isolated peritonealmacrophages from LPLA2−/− mice are characterized by the inability todigest apoptotic thymocytes but normal capacity to bind and endocytoseapoptotic thymocytes. The finding that macrophages from LPLA2 null micefail to digest engulfed apoptotic bodies indicates that this impairmentis due to the absence of LPLA2. Treatment with exogenous LPLA2 isexpected to increase digestion and clearance of apoptotic bodies, and toreduce accumulation of tingible body macrophages.

The data also show that cationic amphiphilic drugs inhibit LPLA2activity and result in cellular phospholipidosis. The mechanismunderlying this inhibition appears to be interference with theelectrostatic interactions between LPLA2 and anionic lipid membranes.

Finally, the data also demonstrate that mRNA expression of LPLA2 isdecreased in the microdissected glomeruli of patients with lupus,compared to the glomeruli of normal controls and glomeruli of patientswith other glomerular diseases resulting from diabetes, hypertensivenephrosclerosis, minimal change disease, and IgA nephropathy.

Tingible Body Macrophages

Tingible body macrophages are large, mononuclear phagocytic cells foundin the germinal centers of lymphatic tissue (Smith et al., DevelopmentalImmunology 6(3-4): 285-294, 1998). These macrophages contain manyphagocytized, apoptotic cells (referred to as tingible bodies) invarious states of degradation (Swartzendruber and Congdon, Journal ofCell Biology 19: 641-646, 1963; Tabe et al, Acta OtolaryngologicaSupplement 523: 64-67, 1996). These cells have been referred to astype-1 phagocytes. (Kotani et al., Acta Anatomica (Basel) 99(4):391-402, 1977)

These macrophages sometimes range in size from 20-30 μm or larger andcontain a variable number of inclusions. The inclusions represent notonly nuclear, but also cytoplasmic debris in varying stages of lysis.Various cell types form the debris constituting tingible bodies. Forexample, plasmacyte and lymphocyte debris, granulocytes as well asphagocytized erythrocytes have been identified in macrophages fromgerminal centers. (Swartzenbruber et al., J. Cell Biology, 19:641-646,1963)

One of the earliest changes in apoptosis is the exposure ofphosphatidylserine (PS) and phosphatidylethanolamine (PE) on the outerleaflet of the plasma membrane. Tingible body macrophages stronglyexpress milk fat globule epidermal growth factor-8 (MFG-E8) which bindsto apoptotic cells by recognizing PS and enhances the engulfment ofapoptotic cells. Knock-out mice lacking expression of MFG-E8 carry manyunengulfed apoptotic cells in the germinal centers of the spleen, anddevelop a lupus-like autoimmune disease (Hanayama et al., Science304(5674): 1147-1150, 2004 and Current Directions in Autoimmunity 9:162-172, 2006). Apoptotic bodies are characterized by intact plasmamembranes and expression of PS on the outer leaflet of the plasmamembrane.

Increased accumulation of tangible body macrophages is found lymph nodegerminal centers of rheumatoid arthritis patients (Imai et al., ActaPathol Jpn. 1989 February; 39(2):127-34).

SLE and Clearance of Apoptotic Cells

The failed or reduced clearance of apoptotic cells has emerged as one ofthe most popular hypotheses for the development of autoimmunity (Erwiget al., Am J Pathol 171: 2-8, 2007; Savill et al., Nature 407: 784-788,2000; Tanaka et al., Curr Med Chem 14: 2892-2897, 2007). The clearanceof apoptotic cells by phagocytosis can be divided into four distinctsteps. These steps include the recruitment of phagocytes to the siteswhere apoptotic cells are located, the recognition of apoptotic cellsthrough receptors and bridging molecules, the endocytosis of apoptoticbodies into phagocytes, and the digestion of endocytosed cell bodies.While a great deal of progress has been made in delineating themechanisms associated with recognition and endocytosis, very little hasbeen elucidated with regard to the digestion of apoptotic bodies thathave been endocytosed.

There are three characteristic features of lupus that may reflect theimpairment of apoptotic cell clearance. These pathogenomic featuresinclude the lupus erythematosus (LE) cell (Hargraves et al., Mayo ClinProc 23: 25-28, 1948), hematoxylin bodies (Wilson et al, Am J Med Sci241: 31-43, 1961), and tingible body macrophages (Baumann et al.,Arthritis Rheum 46: 191-201, 2002). What little that has been publishedon the ultrastructural characterization of these pathologicalabnormalities suggests that they represent the persistent presence ofapoptotic bodies ingested by phagocytic cells in the bone marrow, kidneyand lung, and lymphoid tissues respectively (Ruiz-Arguelles et al.,Scand J Clin Lab Invest Suppl 235: 31-37, 2001; Swartzendruber et al., JCell Biol 19: 641-646, 1963). Baumann et al. (2002) have reported thatapoptotic cells are not properly cleared by tingible body macrophages ofthe germinal centers in a sub-group of patients with SLE. (Baumann etal., Arthritis and Rheumatism 46(1): 191-201, 2002).

SLE includes systemic lupus erythematosus or any other autoimmunedisease displaying SLE symptoms or accumulation of tingible bodymacrophages. Autoantibodies play a role in disease development in SLE.(Rahman, Rheumatology 43(11):1326-1336, 2004; Ehrenstein, Rheumatology38: 691-693, 1999). Targets of autoantibodies in systemic lupuserythematosus include nuclear and cytoplasmic macromolecules, lipidcomponents and plasma proteins. Autoantibodies are involved in diseasedevelopment either by forming immune complexes that lodge in targetorgans, disrupting normal organ function, or by cross-reacting withtargeted antigens and damaging tissue. The most frequently associatedautoantibodies in systemic lupus erythematosis include antibodies toSmith (a ribonucleoprotein), and antibodies to nucleosomes, histones,and double stranded (ds) DNA. Anti-ds DNA antibodies are the mostfrequently detected antibodies in SLE. Titers of dsDNA antibodies can beused to diagnose systemic lupus and evaluate disease activity.

Systemic lupus erythematosus is a multisystem disease. The most commoninitial signs of the disease are fatigue, fever, and muscle and jointpain. Muscle pain (myalgia), joint pain (arthralgia) and arthritis arecommon with the new onset of lupus and with subsequent flare-ups. Themuscles themselves can sometimes become inflamed and very painfulcontributing to weakness and fatigue. The most frequent joints involvedin lupus arthritis are those of the hand, knees, and wrists.Complications caused by reduced blood flow to a joint can cause death ofthe bone in the joint (avascular necrosis). Avascular necrosis occursmost commonly in the hips and knees.

Skin symptoms occur in more than 90% of patients with lupus and caninclude many different types of rashes. The classic lupus rash is aredness on the cheeks (malar blush) often brought on by sun exposure.Discoid lupus with red skin patches on the skin and scaliness is acharacteristic rash that can lead to scarring. It usually occurs on theface and scalp and can lead to loss of scalp hair (alopecia).

Kidney complications, glomerulonephritis due to deposition of immunecomplexes in the kidney, are observed in more than half of all patientswith lupus. Severe kidney disease often requires immunosuppressivetherapy. More than 50% of patients with lupus have lung complications.Inflammation of the lining of the lung (pleurisy) is common. Pleuraleffusions can occur as well. Inflammation of the arteries (vasculitis)can occur, as well as inflammation around the heart (pericarditis).Patients with lupus are also more predisposed to developatherosclerosis.

Nervous system involvement occurs in about 15% of patients with lupus.Potential symptoms include seizures, nerve paralysis, severe depression,psychosis, and strokes. Spinal cord inflammation in lupus is rare butcan cause paralysis. About half of patients with lupus have anemiaand/or thrombocytopenia and/or leukopenia. Some lupus patients arepredisposed to developing blood clots. This is most likely to occur inpatients who have antiphospholipid antibodies. Inflammation or infectionof the intestines can occur, due to a blood clot or inflammation ofblood vessels in the intestines. Some patients experience intermittentinterruptions in blood supply to the hand (Raynaud's syndrome), whichmanifests as whiteness, blueness and pain in the fingers.

Drug-induced lupus (DEL) is a disorder with symptoms similar to systemiclupus erythematosus, which is induced by chronic use of certain drugs.Drug-induced lupus is a well-known entity, accounting for 5-10% of alllupus syndromes has been reported as a side-effect of long-term therapywith over 40 medications. (Rubin, Toxicology 209(2):135-47, 2005;Perez-Garcia et al., Rheumatology, 45(1):114-116, 2006). Its clinicaland laboratory features are similar to systemic lupus erythematosus,except that patients generally recover after the offending medication isdiscontinued. Although over 40 medications are known to be associatedwith drug-induced lupus, the three that report the highest numbers are:procainamide (brand name Pronestyl, used to treat heart arrythmias),hydralazine (brand name Apresoline, used to tread hypertension) andquinidine (brand name Quinaglute, used to treat heart arrythmias).(Hofstra, Drug Metab Rev 26 (3):485-505, 1994; Uetrecht et al. Chem ResToxicol 1 (1): 74-8, 1998). Drugs that cause drug-induced lupus includeAcebutolol, Amiodarone, Bupropion, Captopril, Carbamazepine,Chlorpromazine, Diltiazem, Docetaxel, Ethosuximide, Gemfibrozil,Glyburide, Gold salt, Griseofulvin, Hydantoins, Hydralazine,hydroxychloroquine, Interferons, Interleukins, Isoniazid, Leuprolideacetate, Lithium, Lovastatin, Mephenyloin, Methyldopa, Minocycline,Nitrofurantoin, Olanzapine, Ophthalmic timolol, Oral contraceptives,Penicillamine, Phenyloin, Practolol, Procainamide, Propylthiouracil,Quinidine, Reserpine, Rifampin, Simvastatin, Sulfasalazine,Tetracycline, Ticlopidine, Tiotropium bromide inhaler, Trimethadione,Tumor necrosis factor, Valproate, or Voriconazole. Drugs that causeflares of systemic lupus erythematosus are as follows: Cimetidine,Hydralazine, Hydrochlorothiazide, Mesantoin, P-Aminobenzoic acid (PABA),Penicillin, Phenylbutazone, Sulfonamides, or Terbinafine.

Lysosomal Phospholipase A2 and the Phospholipase Superfamily

The present section provides a general description of the family ofphospholipases, and LPLA2 enzymes specifically.

The phospholipase A2 superfamily is comprised of a broad range ofenzymes that share the ability to hydrolyze the sn-2 ester bond ofphospholipids. The products of this reaction, free fatty acid andlysophospholipid, have important biological roles. The former is notonly an important source of cellular energy, but is also the substratefor additional cellular messengers in the form of arachidonatemetabolites. The latter products play signaling roles in addition tohaving important effects on membrane remodeling and membraneperturbation. Historically the PLA2s were thought to be small, secretedenzymes characterized by having a catalytic histidine, calciumdependence and being disulfide rich. The family of enzymes greatlyexpanded with the discovery of cytosolic PLA2 activity assignable to aprotein that lacked disulfide bonds and characterized by presence of acatalytic serine (Gronich et al., J Biol Chem 263: 16645-16651, 1988).Currently there are fifteen groups of phospholipase A2s. Groups I, II,II, V, IX, X, and XI utilize a catalytic histidine and are of smallmolecular weight (13-15 kDa). Groups IV, VI, VII, and VIII utilize anucleophilic serine and have little or no Ca2+ requirement forcatalysis. The molecular weights are larger (26-114 kDa). Many membersof these latter groups have C2 domains, are members of the α/β□ foldhydrolase proteins, and may contain a required Ser/His/Asp triad foractivity.

Lysosomal phospholipase A2 (LPLA2, LLPL, LYPLA3), also known as1-O-acylceramide synthase, was recently recognized as the first memberof a new group XV of PLA2s (Schaloske et al., Biochim Biophys Acta 1761:1246-1259, 2006). LPLA2 is a single-chain, mannose-rich glycoproteinhaving a molecular mass of about 40 kDa. LPLA2 has both lipase andtransacylase activities, and an acidic pH optimum. It is specific forthe phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine(PE). The protein is selectively and highly expressed in alveolarmacrophages but is also present to a lesser degree in peritonealmacrophages, peripheral blood monocytes, or other tissues. Othermacrophage-associated phospholipase A2s do not show a comparabledistribution.

LPLA2 is secreted from macrophages and mast cells in response toagonists. LPLA2 is recognized by cellular mannose receptors and isreincorporated and trafficked back to the lysosome when exogenouslyadministered. The secretion of LPLA2 results in the formation ofbioactive lipid ligands with known effects on lymphocyte recruitment,proliferation, and cytokine production.

In 1996, the existence of a novel pathway for ceramide metabolism wasreported (Abe, A., Shayman, J. A., and Radin, N. S. J Biol Chem 271,14383-14389, 1996). This pathway results in the formation of a highlylipophilic metabolite of ceramide, 1-O-acylceramide (FIG. 1A). Underthis reaction, a fatty acid from the sn-2 position ofphosphatidylcholine or phosphatidylethanolamine is transferred to theC-1 hydroxyl group of ceramide. Upon purification of the enzyme, it wasdetermined that either ceramide or water could serve as acceptors forsn-2 fatty acid (Abe, A., and Shayman, J. A. J Biol Chem 273, 8467-8474,1998). In the presence of ceramide, LPLA2 catalyzes the formation of1-O-acylceramide by transacylation of fatty acids from the sn-2 positionof phosphatidylcholine or phosphatidylethanolamine. When water serves asthe acceptor, in the absence of ceramide or other alcohols, the enzymeacts as a traditional phospholipase A2. Two characteristics of thisphospholipase A2 immediately became apparent. First, the enzymedisplayed acidic pH optimum, about 4.5; second, the enzyme localized tolysosomes.

Based on the enzyme purification, several partial amino acid sequencesof LPLA2 were determined and the full length of coding and full lengthamino acid sequences determined (Hiraoka et al., J Biol Chem 277,10090-10099, 2002). The expressed protein was confirmed to have bothphospholipase A2 and transacylase activities. Analysis of the codingsequence revealed LPLA2 is 49% identical at the amino acid level withlecithin cholesterol acyltransferase (LCAT). Most of the identity waslocalized to the catalytic domain (FIG. 1B). Both LCAT and LPLA2colocalized to 16q22 where they are contiguously placed, consistent withone gene arising from a duplication event. Phylogenetic analysisrevealed that both genes were derived from diacylglycerolacyltransferase and that LPLA2 is ancestral to LCAT.

The sequence of human LPLA2 is set forth in SEQ ID NO: 1. A moredetailed analysis of the catalytic domain revealed that LPLA2 contains aconsensus sequence, -A-X-S-X-G- (amino acids 196-200 of SEQ ID NO: 1),present in other phospholipase A2s and acylhydrolases. The serine ispart of a catalytic triad composed of an asparagine and histidineresidue in close approximation and serving to convert the serine into astrong nucleophile. Site directed mutagenesis of the triad amino acidseliminated the intrinsic phospholipase and transacylase activities ofLPLA2. Additional analysis revealed the presence of a single disulfidebridge between Cys65 and Cys89 (Hiraoka, M., Abe, A., and Shayman, J. A.J Lipid Res 46, 2441-2447, 2005). More recently, the sn-1 versus sn-2specificity of the enzyme has been assessed. LPLA2 displays highspecificity toward phospholipids containing arachidonate in the sn-2position. However, both sn-1 and sn-2 hydrolase activities are seen whenother fatty acyl substitutions are present (Abe, A., Hiraoka, M., andShayman, J. A. J Lipid Res 47, 2268-2279, 2006). Other lipophilicalcohols besides ceramide can serve as acceptors for the fatty acid viathe transacylation activity (Abe, A., Hiraoka, M., and Shayman, J. A. JLipid Res 48, 2255-2263, 2007).

The divalent cations Ca2+ and Mg2+ enhance, but are not required for,LPLA2 transacylase activity. LPLA2 is neither activated nor inhibited inthe presence of ATP or thiol reagents such as dithiothreitol and NEM.Thus the enzyme differs from groups I, II, and III phospholipase A2s,which are highly sensitive to such reagents. The phospholipase A2inhibitors bromoenollactone (BEL) and nonadecyltetraenyl trifluoromethylketone (AACOF3) do not inhibit the enzyme activity.

LPLA2 is a high mannose-type glycoprotein, suggesting that the releasedenzyme might be reincorporated into macrophages via a mannose ormannose-6-phosphate receptor. Recombinant glycosylated mouse LPLA2produced by HEK293 cells was applied to LPLA2-deficient (LPLA2−/−) mousealveolar macrophages. The uptake of exogenous LPLA2 into LPLA2−/−alveolar macrophages occurred in a concentration-dependent manner andcolocalized with the lysosomal marker, Lamp-1. (Abe et al. J. Immunol,181(11):7873-81, 2008) This uptake was significantly suppressed in thepresence of alpha-methyl-mannoside but not in the presence of mannose6-phosphate. Thus, the predominant pathway for uptake of exogenous LPLA2is via the mannose receptor. (Abe et al., J. Immunol, 181(11):7873-81,2008).

LPLA2 Fragments, Variants and Derivatives

LPLA2 that are useful according to the methods of the invention maycomprise an amino acid sequence of any one of SEQ ID NOs: 1-288, orbiologically active fragments or variants or derivatives thereof.Compositions comprising such LPLA2, fragments, variants or derivativesare used in the treatment of disorders involving impaired clearance ofapoptotic bodies and/or accumulation of tingible body macrophages.Polynucleotides encoding the LPLA2, fragments or variants thereof, arealso useful in the present invention, e.g. for gene therapy purposes orrecombinant production of the protein.

LPLA2 proteins include any mammalian protein that comprises the aminoacid sequences of SEQ ID NOs: 1-288, a fragment of SEQ ID NOs: 1-288, ora variant or conservative substitution variant of a protein of SEQ IDNOs: 1-288, or a derivative thereof that retains the desired biologicalactivity. In certain aspects, the LPLA2 protein is derived from anynatural source, e.g., a mammalian origin such as human (SEQ ID NO: 1),bovine, murine (e.g., of these sequences are depicted in FIG. 1C andHiraoka et al., J Biol Chem 277, 10090-9, 2002), or alternatively, it isproduced synthetically or through recombinant methods known to those ofskill in the art. For the purposes of treating disorders associated withaccumulation of tingible body macrophages, e.g., SLE or systemic lupuserythematosus, the desired retained biological activity is a cataboliceffect on phospholipids, and/or improved clearance of apoptotic bodiesand/or reduced accumulation of tingible body macrophages.

A. Fragments and Variants

Truncation of amino acids at the N-terminus or C-terminus of any of SEQID NO: 1-288 may provide fragments that retains the desired biologicalactivity. Fragments of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, or 150 consecutive amino acids are contemplated.Such fragments preferably comprise the catalytic consensus sequence,-A-X-S-X-G-, corresponding to amino acids 196-200 of SEQ ID NO: 1. Suchfragments may also include a cysteine bond corresponding to the cysteinebond between the Cys at position 65 and the Cys 89 of SEQ ID NO: 1.

The term “variant” is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore sites in the native protein and/or substitution of one or moreamino acids at one or more sites in the native protein. Variants arereadily made by any of the variety of means known in the art, includingthrough recombinant production. Polynucleotides encoding variants may bemade, e.g. through site-directed mutagenesis of polynucleotides encodingLPLA2, or through direct synthesis of the encoding polynucleotidesequence.

Biologically active variants of a native LPLA2 protein of the inventionwill have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to the amino acid sequence for the native protein as determinedby sequence alignment programs. A biologically active variant of aprotein of the invention may differ from that protein by as few as 1-15amino acid residues, as few as 1-10, such as 6-10, as few as 5, as fewas 4, 3, 2 or even 1 amino acid residue. Biologically active variantsalternatively are encoded by a polynucleotide that hybridizes understringent conditions to the complement of a polynucleotide that encodesSEQ ID NO: 1. Highly stringent conditions include hybridization and washconditions of relatively low ionic strength and high temperature.Exemplary highly stringent wash conditions comprise washing in 0.1×SSCat 65-68° C. Exemplary moderately stringent wash conditions comprisewashing in 1×SSC at 55° C.

As another example, conservative substitution or non-conservativesubstitution, insertion or deletion of amino acid residues of any of SEQID NO: 1-288 may produce variants that retain the desired biologicalactivity and/or retain three-dimensional conformation structure of theprotein of SEQ ID NOs: 1-288. Such variants preferably comprise thecatalytic consensus sequence, -A-X-S-X-G-. Such variants may alsoinclude a cysteine bond corresponding to the cysteine bond between theCys at position 65 and the Cys 89 of SEQ ID NO: 1.

The term “conservative substitution” as used herein denotes thereplacement of an amino acid residue by another, biologically similarresidue with respect to hydrophobicity, hydrophilicity, cationic charge,anionic charge, shape, polarity and the like. Examples of conservativesubstitutions include the substitution of one hydrophobic residue suchas isoleucine, valine, leucine, alanine, cysteine, glycine,phenylalanine, proline, tryptophan, tyrosine, norleucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic acid for asparticacid, or glutamine for asparagine, and the like. Neutral hydrophilicamino acids which are substituted for one another include asparagine,glutamine, serine and threonine. The term “conservative substitution”also includes the use of a substituted or modified amino acid in placeof an unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide. By “substituted” or “modified” the present inventionincludes those amino acids that have been altered or modified fromnaturally occurring amino acids.

As such, it should be understood that in the context of the presentinvention, a conservative substitution is recognized in the art as asubstitution of one amino acid for another amino acid that has similarproperties. Exemplary conservative substitutions are set out in e.g.,Alternatively, conservative amino acids are grouped as described inLehninger, (Biochemistry, Second Edition; Worth Publishers, Inc.N.Y.:N.Y. (1975), pp. 71-77). Those of skill in the art are aware ofnumerous tables that indicate specific conservative substitutions. Oneexemplary such table is provided below:

Table of Exemplary Conservative Substitutions Original Residue ExemplarySubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

Any variant of a protein of SEQ ID NOs: 1-288 that retains most or allof the catalytic domain of the LPLA2 of SEQ ID NOs: 1-288 iscontemplated to be useful in the methods of the present invention. Asdescribed above, the catalytic domain of LPLA2 contains the consensussequence, -A-X-S-X-G-, present in other phospholipase A2s andacylhydrolases. The serine is part of a catalytic triad composed of anasparagine and histidine residue in close approximation and serving toconvert the serine into a strong nucleophile. One approach to makingvariants is to begin with conservative substitutions within or near thecatalytic domain, while retaining the consensus catalytic sequence,followed by conservative substitutions or non-conservativesubstitutions, insertions or deletions outside of the catalytic domain.LPLA2 of SEQ ID NO: 1 has 50% homology to cholesterol lecithinacyltransferase (LCAT), and the majority of this homology is within thecatalytic domain. Thus, it is contemplated that those of skill in theart may choose to produce variants of SEQ ID NO: 1 in which thecatalytic domain of SEQ ID NO: 1 is replaced by the catalytic domain ofan LCAT (Hiraoka et al., J Biol Chem 277, 10090-9, 2002), as long assuch a variant retains its property of catalyzing phospholipidbreakdown. Such activities are readily assessed as described hereinbelow.

In addition, rational drug design is used to produce structural variantsof the LPLA2 proteins and thus provide additional compositions for usein the methods contemplated herein. By creating such variants, theskilled worker can fashion LPLA2-derived proteins which are more activeor stable than the natural molecules which have different susceptibilityto alteration or which may affect the function of various othermolecules. In one approach, it is desirable to generate athree-dimensional structure for LPLA2-derived protein of interest or afragment thereof e.g., by x-ray crystallography, computer modeling or bya combination of both approaches. An alternative approach, “alaninescan,” involves systematic replacement of residues throughout theprotein with alanine, followed by determining the resulting effect onfunction.

Furthermore, nonpeptide variants of LPLA2-derived proteins that providea stabilized structure or lessened biodegradation, are alsocontemplated. Peptide mimetic variants are prepared based on theunderlying LPLA2 protein structure by replacing one or more amino acidresidues of the protein of interest by nonpeptide moieties. In oneaspect, the nonpeptide moieties permit the peptide to retain its naturalconfirmation, or stabilize a bioactive confirmation. One example ofmethods for preparation of nonpeptide mimetic variants from peptides isdescribed in Nachman et al., Regul. Pept. 57:359-370 (1995). “Peptide”as used herein embraces all of the foregoing.

B. Derivatives

In addition to the basic amino acid structure of the proteins, it iscontemplated that the LPLA2 proteins will be modified to enhance theiruptake, decrease toxicity, increase circulatory time, or modifybiodistribution of the LPLA2 proteins. For example, any modificationthat facilitates the greater uptake of LPLA2 compositions bymacrophages, e.g. modifications that increase mannose residues, arecontemplated. Mannose content of LPLA2 can be increased, for example, bychemical conjugation of mannose, by treatment with enzymes that exposeor add mannose to glycosylation sites, or by modification of recombinantproduction methods to favor high mannose content of the resultingglycoprotein. Compositions of LPLA2 with high mannose content canalternatively be prepared by affinity purification using a matrixcoupled to mannose binding protein, e.g. a mannose receptor, amannose-binding lectin, and the like.

The compounds of the invention may also be covalently or noncovalentlyassociated with a carrier molecule, such as a linear polymer (e.g.,polyethylene glycol, polylysine, dextran, etc.), a branched-chainpolymer (see, for example, U.S. Pat. No. 4,289,872; U.S. Pat. No.5,229,490; WO 93/21259); a lipid; a cholesterol group (such as asteroid); or a carbohydrate or oligosaccharide. Other possible carriersinclude antibody moieties, and in particular constant regions derivedfrom an antibody. Still other possible carriers include one or morewater soluble polymer attachments such as polyoxyethylene glycol, orpolypropylene glycol as described U.S. Pat. Nos. 4,640,835, 4,496,689,4,301,144, 4,670,417, 4,791,192 and 4,179,337. Still other usefulpolymers known in the art include monomethoxy-polyethylene glycol,dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol, as well as mixtures of thesepolymers.

For example, an Fc region can be fused to the N-terminus or C-terminus(or both) of the LPLA2 protein, fragment or variant. Multiple vehicles,as exemplified herein, may also be used; e.g., an Fc at a terminus and aPEG group at the other terminus or a side chain.

In various embodiments, the Fc component is either a native Fc or an Fcvariant. By way of example and without limitation, the Fc component isan Fc region of the human immunoglobulin IgG1 heavy chain or abiologically active fragment, derivative, or dimer thereof, see Ellison,J. W. et al., Nucleic Acids Res. 10:4071-4079 (1982). It is understood,however, that an Fc region for use in the invention may be derived froman IgG, IgA, IgM, IgE or IgD from any species. Native Fc domains aremade up of monomeric polypeptides that may be linked into dimeric ormultimeric forms by covalent (i.e., disulfide bonds) and/or non-covalentassociation. The number of intermolecular disulfide bonds betweenmonomeric subunits of native Fc molecules ranges from 1 to 4 dependingon class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3,IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimerresulting from papain digestion of an IgG (see Ellison et al. (1982),Nucleic Acids Res. 10: 4071-9).

Various water-soluble polymers have been shown to modifybiodistribution, improve the mode of cellular uptake, change thepermeability through physiological barriers, and modify the rate ofclearance from the body. (Greenwald et al., Crit Rev Therap Drug CarrierSyst. 2000; 17:101-161; Kopecek et al., J Controlled Release.,74:147-158, 2001). To achieve either a targeting or sustained-releaseeffect, water-soluble polymers have been synthesized that contain drugmoieties as terminal groups, as part of the backbone, or as pendentgroups on the polymer chain.

Polyethylene glycol (PEG), has been widely used as a drug carrier, givenits high degree of biocompatibility and ease of modification. Harris etal., Clin Pharmacokinet. 2001; 40(7):539-51 Attachment to various drugs,proteins, and liposomes has been shown to improve residence time anddecrease toxicity. (Greenwald et al., Crit Rev Therap Drug Carrier Syst.2000; 17:101-161; Zalipsky et al., Bioconjug Chem. 1997; 8:111-118). Inone aspect, PEG is coupled to active agents through the hydroxyl groupsat the ends of the chain and via other chemical methods; however, PEGitself is limited to at most two active agents per molecule. In adifferent approach, copolymers of PEG and amino acids were explored asbiomaterials which retain the biocompatibility properties of PEG, butwhich have the added advantage of numerous attachment points permolecule (providing greater drug loading), and which could besynthetically designed to suit a variety of applications (Nathan et al.,Macromolecules. 1992; 25:4476-4484; Nathan et al., Bioconj Chem. 1993;4:54-62).

Those of skill in the art are aware of PEGylation techniques for theeffective modification of drugs. For example, drug delivery polymersthat consist of alternating polymers of PEG and tri-functional monomerssuch as lysine have been used by VectraMed (Plainsboro, N.J.). The PEGchains (typically 2000 daltons or less) are linked to the α- and ε-aminogroups of lysine through stable urethane linkages. Such copolymersretain the desirable properties of PEG, while providing reactive pendentgroups (the carboxylic acid groups of lysine) at strictly controlled andpredetermined intervals along the polymer chain. In one aspect, thereactive pendent groups are used for derivatization, cross-linking, orconjugation with other molecules. These polymers are useful in producingstable, long-circulating pro-drugs by varying the molecular weight ofthe polymer, the molecular weight of the PEG segments, and the cleavablelinkage between the drug and the polymer. The molecular weight of thePEG segments affects the spacing of the drug/linking group complex andthe amount of drug per molecular weight of conjugate (smaller PEGsegments provides greater drug loading). In general, increasing theoverall molecular weight of the block co-polymer conjugate increases thecirculatory half-life of the conjugate. Nevertheless, the conjugate musteither be readily degradable or have a molecular weight below thethreshold-limiting glomular filtration (e.g., less than 45 kDa). Thus,in one aspect, PEGylated LPLA2 proteins are in the range of between 20and 35 kDa in molecular weight.

Another set of useful derivatives are the LPLA2 proteins conjugated toother therapeutic agents or diagnostic agents, including tracers, orradioisotopes. Useful conjugation partners include: radioisotopes, suchas 90Yttrium, 131Iodine, 225Actinium, and 213Bismuth; ricin A toxin,microbially derived toxins such as Pseudomonas endotoxin (e.g., PE38,PE40), and the like; partner molecules in capture systems (see below);biotin, streptavidin (useful as either partner molecules in capturesystems or as tracers, especially for diagnostic use); andchemotherapeutic agents.

Methods of Faking LPLA2 Proteins

LPLA2 proteins can be produced by conventional automated peptidesynthesis methods or by recombinant expression. General principles fordesigning and making proteins are well known to those of skill in theart.

A. Automated Solid-Phase Peptide Synthesis

In one aspect any protein of the invention is synthesized in solution oron a solid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and is used inaccordance with known protocols. See, for example, Stewart and Young,Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co., (1984); Tamet al., J. Am. Chem. Soc., 105:6442, (1983); Merrifield, Science, 232:341-347, (1986); and Barany and Merrifield, The Peptides, Gross andMeienhofer, eds, Academic Press, New York, 1-284, (1979), eachincorporated herein by reference. As such, LPLA2 proteins, fragments andvariants thereof are readily synthesized and optionally screened for arelated activity e.g., aclyceramide synthase activity assays.

For example, the peptides are synthesized by solid-phase technologyemploying an exemplary peptide synthesizer such as a Model 433A fromApplied Biosystems Inc. In such cases, the purity of any given peptidesubstrate, generated through automated peptide synthesis or throughrecombinant methods, is typically determined using reverse phase HPLCanalysis. Chemical authenticity of each peptide is established by anymethod well known to those of skill in the art. In certain embodiments,the authenticity is established by mass spectrometry. Additionally, thepeptides also are quantified using amino acid analysis in whichmicrowave hydrolyses are conducted, e.g. using a microwave oven such asthe CEM Corporation's MDS 2000 microwave oven. In certain aspects, thesamples are analyzed by reverse phase HPLC and quantification isachieved using an enhanced integrator. Those of skill in the art arereferred to Hiraoka et al., which describes details of methods ofdetermining amino acid sequence of LPLA2 using a combination of reversephase HPLC and mass spectrometry. Such methods are well known to thoseof skill in the art and are readily adapted for the sequence analysis ofany protein or peptide.

B. Recombinant Protein Production

As an alternative to automated peptide synthesis, recombinant DNAtechnology is employed wherein a nucleotide sequence which encodes apeptide of the invention is inserted into an expression vector,transformed or transfected into an appropriate host cell and cultivatedunder conditions suitable for expression as described herein below. Inone aspect, a nucleotide sequence that encodes a protein of SEQ ID NOs:1-288, a fragment or variant thereof is provided. Recombinant methodsare especially useful for producing longer polypeptides for use in themethods of the invention.

A variety of expression vector/host systems are known in the art. Theseinclude but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti orpBR322 plasmid); or animal cell systems. Mammalian cells that are usefulin recombinant protein productions include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells(such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and293 cells. Exemplary protocols for the recombinant expression of theLPLA2 polypeptides in bacteria, yeast and other invertebrates are knownto those of skill in the art and briefly described below.

Expression vectors for use in prokaryotic hosts generally comprise oneor more phenotypic selectable marker genes. Such genes generally encode,e.g., a protein that confers antibiotic resistance or that supplies anauxotrophic requirement. A wide variety of such vectors are readilyavailable from commercial sources. Examples include pSPORT vectors, pGEMvectors (Promega), pPROEX vectors (LTI, Bethesda, Md.), Bluescriptvectors (Stratagene), pET vectors (Novagen) and pQE vectors (Qiagen).

The DNA sequence encoding the given protein or fusion polypeptide isamplified by PCR and cloned into a vector, for example,pGEX-3×(Pharmacia, Piscataway, N.J.) designed to produce a fusionprotein comprising glutathione-S-transferase (GST), encoded by thevector, and a protein encoded by a DNA fragment inserted into thevector's cloning site. Typically, the primers for the PCR are generatedto include for example, an appropriate cleavage site. Plasmid DNA fromindividual transformants is purified and partially sequenced using anautomated sequencer to confirm the presence of the desired encodingnucleic acid insert in the proper orientation. The vector is transformedinto cells and the LPLA2 protein of interest is purified and recoveredby cleavage of the recombinant fusion protein with thrombin or factor Xa(Pharmacia, Piscataway, N.J.).

The secreted recombinant protein is purified from the bacterial culturemedia by conventional protein purification methods. Similar systems forthe production of recombinant protein in yeast host cells are readilycommercially available, e.g., the Pichia Expression System (Invitrogen,San Diego, Calif.), following the manufacturer's instructions. Anotheralternative recombinant production is achieved using an insect system.Insect systems for protein expression are well known to those of skillin the art. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The substratecoding sequence is cloned into a nonessential region of the virus, suchas the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of substrate will render the polyhedringene inactive and produce recombinant virus lacking coat protein coat.The recombinant viruses are then used to infect S. frugiperda cells orTrichoplusia larvae in which the substrate is expressed (Smith et al., JVirol 46: 584, 1983; Engelhard E K et al., Proc Nat Acad Sci 91: 3224-7,1994).

Mammalian host systems for the expression of recombinant proteins alsoare well known to those of skill in the art. Host cell strains aretypically chosen for a particular ability to process the expressedprotein or produce certain post-translation modifications that will beuseful in providing protein activity. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, andthe like, have specific cellular machinery and characteristic mechanismsfor such post-translational activities and are chosen to ensure thecorrect modification and processing of the introduced, foreign protein.

In one aspect, the transformed cells are used for long-term, high-yieldprotein production and as such stable expression is desirable. Once suchcells are transformed with vectors that contain selectable markers alongwith the desired expression cassette, the cells are allowed to grow for1-2 days in an enriched media before they are switched to selectivemedia. The selectable marker is designed to confer resistance toselection and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clumps ofstably transformed cells are proliferated using tissue culturetechniques appropriate to the cell.

A number of selection systems are useful to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. In other aspects, anti-metabolite resistance is used asthe basis of selection for dhfr, which confers resistance tomethotrexate; gpt, which confers resistance to mycophenolic acid; neo,which confers resistance to the aminoglycoside G418; als which confersresistance to chlorsulfuron; and hygro, which confers resistance tohygromycin. Additional selectable genes that are useful include trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine. Markersthat give a visual indication for identification of transformantsinclude anthocyanins, β-glucuronidase and its substrate, GUS, andluciferase and its substrate, luciferin.

C. Expression Constructs for Recombinant Protein Production

Recombinant production of the LPLA2 proteins of the invention employsvectors comprising polynucleotide molecules that encode LPLA2 proteins.Methods of preparing such vectors as well as producing host cellstransformed with such vectors are well known to those skilled in theart. In one aspect, the polynucleotide molecules used (e.g., apolynucleotide encoding a polypeptide of SEQ ID NOs: 1-288 or a fragmentor variant thereof) are joined to a vector, which generally includes aselectable marker and an origin of replication, for propagation in ahost. These elements of the expression constructs are well known tothose of skill in the art. Generally, the expression vectors include DNAencoding the given protein being operably linked to suitabletranscriptional or translational regulatory sequences, such as thosederived from a mammalian, microbial, viral, or insect gene. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, mRNA ribosomal binding sites, and appropriate sequences whichcontrol transcription and translation.

The terms “expression vector,” “expression construct” or “expressioncassette” are used interchangeably throughout this specification and aremeant to include any type of genetic construct containing a nucleic acidcoding for a gene product in which part or all of the nucleic acidencoding sequence is capable of being transcribed, operably linked to aregulatory sequence.

The choice of a suitable expression vector for expression of thepeptides or polypeptides of the invention will of course depend upon thespecific host cell to be used, and is within the skill of the ordinaryartisan. Methods for the construction of mammalian expression vectorsare disclosed, for example, in Okayama and Berg (Mol. Cell. Biol. 3:280(1983)); Cosman et al. (Mol. Immunol. 23:935 (1986)); Cosman et al.(Nature 312:768 (1984)); EP-A-0367566; and WO 91/18982.

In one aspect, expression construct comprises a selectable marker thatallows for the detection of the expression of a peptide or polypeptide.Usually the inclusion of a drug selection marker aids in cloning and inthe selection of transformants, for example, neomycin, puromycin,hygromycin, DHFR, zeocin and histidinol. Alternatively aspects employenzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic),β-galactosidase, luciferase, or chloramphenicol acetyltransferase (CAT)(prokaryotic) as markers. Alternatively, immunologic markers also areemployed. For example, epitope tags such as the FLAG system (IBI, NewHaven, Conn.), HA and the 6×His system (Qiagen, Chatsworth, Calif.) areemployed. Additionally, glutathione S-transferase (GST) system(Pharmacia, Piscataway, N.J.), or the maltose binding protein system(NEB, Beverley, Mass.) also are used. The selectable marker employed isnot believed to be important, so long as it is capable of beingexpressed simultaneously with the nucleic acid encoding a gene product.Further examples of selectable markers are well known to one of skill inthe art.

Expression requires that appropriate signals be provided in the vectors,such as enhancers/promoters from both viral and mammalian sources thatare used to drive expression of the nucleic acids of interest in hostcells. Usually, the nucleic acid being expressed is undertranscriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. Nucleotide sequences are operably linked whenthe regulatory sequence functionally relates to the DNA encoding thepeptide substrate or the fusion polypeptide. Thus, a promoter nucleotidesequence is operably linked to a given DNA sequence if the promoternucleotide sequence directs the transcription of the sequence.Similarly, the phrase “under transcriptional control” means that thepromoter is in the correct location and orientation in relation to thenucleic acid to control RNA polymerase initiation and expression of thegene.

Any promoter that will drive the expression of the nucleic acid is used.The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. In one aspect, such a promoter includes either a human or viralpromoter. Common promoters include, e.g., the human cytomegalovirus(CMV) immediate early gene promoter, the SV40 early promoter, the Roussarcoma virus long terminal repeat, β-actin, rat insulin promoter, thephosphoglycerol kinase promoter and glyceraldehyde-3-phosphatedehydrogenase promoter, all of which are promoters well known andreadily available to those of skill in the art and are used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter with wellknown properties, the level and pattern of expression of the protein ofinterest following transfection or transformation is optimized.Inducible promoters also are contemplated for use.

Another regulatory element that is used in protein expression is anenhancer. These are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Where an expression construct employs a cDNA insert, one will typicallydesire to include a polyadenylation signal sequence to effect properpolyadenylation of the gene transcript. Any polyadenylation signalsequence recognized by cells of the selected transgenic animal speciesis suitable for the practice of the invention, such as human or bovinegrowth hormone and SV40 polyadenylation signals.

Also contemplated as an element of the expression cassette is aterminator. These elements serve to enhance message levels and tominimize read through from the cassette into other sequences. Thetermination region is selected for convenience, since terminationregions for the applications such as those contemplated by the presentinvention appear to be relatively interchangeable. In certain aspects,the termination region is native with the transcriptional initiation, inother embodiments, it is native to the DNA sequence of interest, oralternatively it is derived for another source.

Gene Therapy

Expression vector can also be used for gene therapy to effect theexpression of the protein in vivo. In one aspect, the expressionconstructs are introduced into the cells targeted for treatment usingany methods known to those of skill in the art. For example, theexpression constructs form part of a viral delivery vector. In otherembodiments, non-viral delivery is contemplated. Receptor-mediateddelivery also is contemplated (Ridgeway, In: Rodriguez R L, Denhardt DT, ed. Vectors: A survey of molecular cloning vectors and their uses.Stoneham: Butterworth, 467 492, 1988; Nicolas and Rubenstein, In:Vectors: A survey of molecular cloning vectors and their uses, Rodriguez& Denhardt (eds.), Stoneham: Butterworth, 493 513, 1988; Baichwal andSugden, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum Press,117 148, 1986; Temin, In: gene Transfer, Kucherlapati (ed.), New York:Plenum Press, 149 188, 1986).

It is now widely recognized that DNA can be introduced into a cell usinga variety of viral vectors. In various embodiments, expressionconstructs comprising viral vectors containing the genes of interest areadenoviral (see for example, U.S. Pat. No. 5,824,544; U.S. Pat. No.5,707,618; U.S. Pat. No. 5,693,509; U.S. Pat. No. 5,670,488; U.S. Pat.No. 5,585,362; each incorporated herein by reference), retroviral (seefor example, U.S. Pat. No. 5,888,502; U.S. Pat. No. 5,830,725; U.S. Pat.No. 5,770,414; U.S. Pat. No. 5,686,278; U.S. Pat. No. 4,861,719 eachincorporated herein by reference), adeno-associated viral (see forexample, U.S. Pat. No. 5,474,935; U.S. Pat. No. 5,139,941; U.S. Pat. No.5,622,856; U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,773,289; U.S. Pat.No. 5,789,390; U.S. Pat. No. 5,834,441; U.S. Pat. No. 5,863,541; U.S.Pat. No. 5,851,521; U.S. Pat. No. 5,252,479 each incorporated herein byreference), an adenoviral-adenoassociated viral hybrid (see for example,U.S. Pat. No. 5,856,152 incorporated herein by reference) or a vacciniaviral or a herpesviral (see for example, U.S. Pat. No. 5,879,934; U.S.Pat. No. 5,849,571; U.S. Pat. No. 5,830,727; U.S. Pat. No. 5,661,033;U.S. Pat. No. 5,328,688 each incorporated herein by reference) vector.

Non-viral gene transfer include calcium phosphate precipitation (Grahamand Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. CellBiol., 7:2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695,1990) DEAE-dextran (Gopal, Mol. Cell Biol., 5:1188-1190, 1985),electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986;Potter et al., Proc. Nat. Acad. Sci. USA, 81:7161-7165, 1984), directmicroinjection (Harland and Weintraub, J. Cell Biol., 101:1094-1099,1985.), DNA-loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta,721:185-190, 1982; Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348-3352, 1979; Felgner, Sci Am. 276(6):102 6, 1997; Felgner, HumGene Ther. 7(15):17913, 1996), cell sonication (Fechheimer et al., Proc.Natl. Acad. Sci. USA, 84:8463-8467, 1987), gene bombardment using highvelocity microprojectiles (Yang et al., Proc. Natl. Acad. Sci. USA,87:9568-9572, 1990), and receptor-mediated transfection (Wu and Wu, J.Biol. Chem., 262:4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892,1988; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993).

Liposomal delivery is also contemplated (Radler et al., Science,275(5301):810 4, 1997). Also contemplated in the present invention arevarious commercial approaches involving “lipofection” technology.Complexing the liposome with a hemagglutinating virus (HVJ) facilitatesfusion with the cell membrane and promotes cell entry ofliposome-encapsulated DNA (Kaneda et al., Science, 243:375-378, 1989).In other exemplary embodiments, the liposome is complexed or employed inconjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Katoet al., J. Biol. Chem., 266:3361-3364, 1991). In yet furtherembodiments, the liposome is complexed or employed in conjunction withboth HVJ and HMG-1. In that such expression constructs have beensuccessfully employed in transfer and expression of nucleic acid invitro and in vivo, then they are applicable for the present invention.

Receptor-mediated gene targeting vehicles also are useful and generallyconsist of two components: a cell receptor-specific ligand and aDNA-binding agent. Several ligands have been used for receptor-mediatedgene transfer. The most extensively characterized ligands areasialoorosomucoid (ASOR) (Wu and Wu, 1987, supra) and transferrin(Wagner et al., Proc. Nat'l. Acad. Sci. USA, 87(9):3410-3414, 1990).Recently, a synthetic neoglycoprotein, which recognizes the samereceptor as ASOR, has been used as a gene delivery vehicle (Ferkol etal., FASEB J., 7:1081-1091, 1993; Perales et al., Proc. Natl. Acad.Sci., USA 91:4086-4090, 1994) and epidermal growth factor (EGF) has alsobeen used to deliver genes to squamous carcinoma cells (Myers, EPO0273085).

In another embodiment of the invention, the expression construct simplyconsists of naked recombinant DNA or plasmids. Transfer of the constructis performed by any of the methods mentioned above which physically orchemically permeabilize the cell membrane. This is applicableparticularly for transfer in vitro, however, it is also applied for invivo use as well. Dubensky et al. (Proc. Nat. Acad. Sci. USA,81:7529-7533, 1984; Benvenisty and Neshif (Proc. Nat. Acad. Sci. USA,83:9551-9555, 1986). Naked DNA expression constructs also aretransferred using particle bombardment (Klein et al., Nature, 327:70-73,1987; Yang et al., Proc. Natl. Acad. Sci. USA, 87:9568-9572, 1990).

Protein Purification

It is desirable to purify the LPLA2 proteins of the invention, forexample, for use in the therapeutic methods of the present invention.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the LPLA2 proteins/polypeptides of the invention from otherproteins, the LPLA2 polypeptides of interest are further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography(FPLC) or even high performance liquid chromatography (HPLC). ExemplaryHPLC conditions include those exemplified in Hiraoka et al., J Biol Chem277, 10090-9, 2002.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedpolypeptide, protein or peptide. The term “purified polypeptide, proteinor peptide” as used herein, is intended to refer to a composition,isolated from other components, wherein the polypeptide, protein orpeptide is purified to any degree relative to its naturally-obtainablestate. A purified polypeptide, protein or peptide therefore also refersto a polypeptide, protein or peptide, free from the environment in whichit may naturally occur.

Generally, “purified” will refer to a polypeptide, protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation refers to a composition in which thepolypeptide, protein or peptide forms the major component of thecomposition, such as constituting about 50%, about 60%, about 70%, about80%, about 90%, about 95% or more of the proteins in the composition.

Various techniques suitable for use in protein purification well knownto those of skill in the art. These include, for example, precipitationwith ammonium sulphate, PEG, antibodies and the like or by heatdenaturation, followed by centrifugation; chromatography steps such asion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps is interchangeable, or that certain steps areomitted, and still result in a suitable method for the preparation of asubstantially purified polypeptide, protein or peptide.

Gene Therapy

Delivery of a therapeutic protein to appropriate cells can be effectedvia gene therapy ex vivo, in situ, or in vivo by use of any suitableapproach known in the art, including by use of physical DNA transfermethods (e.g., liposomes or chemical treatments) or by use of viralvectors (e.g., adenovirus, adeno-associated virus, or a retrovirus). Forexample, for in vivo therapy, a nucleic acid encoding LPLA2, eitheralone or in conjunction with a vector, liposome, or precipitate may beinjected directly into the subject, and in some embodiments, may beinjected at the site where the expression of the LPLA2 compound isdesired. For ex vivo treatment, the subject's cells are removed, thenucleic acid is introduced into these cells, and the modified cells arereturned to the subject either directly or, for example, encapsulatedwithin porous membranes which are implanted into the patient. See, e.g.U.S. Pat. Nos. 4,892,538 and 5,283,187. There are a variety oftechniques available for introducing nucleic acids into viable cells.The techniques vary depending upon whether the nucleic acid istransferred into cultured cells in vitro, or in vivo in the cells of theintended host. Techniques suitable for the transfer of nucleic acid intomammalian cells in vitro include the use of liposomes, electroporation,microinjection, cell fusion, DEAE-dextran, and calcium phosphateprecipitation. A commonly used vector for ex vivo delivery of a nucleicacid is a retrovirus.

Other in vivo nucleic acid transfer techniques include transfection withviral vectors (such as adenovirus, Herpes simplex I virus, oradeno-associated virus) and lipid-based systems. The nucleic acid andtransfection agent are optionally associated with a microparticle.Exemplary transfection agents include calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,quaternary ammonium amphiphile DOTMA ((dioleoyloxypropyl)trimethylammonium bromide, commercialized as Lipofectin byGIBCO-BRL))(Felgner et al, (1987) Proc. Natl. Acad. Sci. USA 84,7413-7417; Malone et al. (1989) Proc. Natl. Acad. Sci. USA 866077-6081); lipophilic glutamate diesters with pendent trimethylammoniumheads (Ito et al. (1990) Biochem. Biophys. Acta 1023, 124-132); themetabolizable parent lipids such as the cationic lipid dioctadecylamidoglycylspermine (DOGS, Transfectam, Promega) and dipalmitoylphosphatidylethanolamylspermine (DPPES)(J. P. Behr (1986) Tetrahedron Lett. 27,5861-5864; J. P. Behr et al. (1989) Proc. Natl. Acad. Sci. USA 86,6982-6986); metabolizable quaternary ammonium salts (DOTB,N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate(DOTAP)(Boehringer Mannheim), polyethyleneimine (PEI), dioleoyl esters,ChoTB, ChoSC, DOSC)(Leventis et al. (1990) Biochim. Inter. 22, 235-241);3beta[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol),dioleoylphosphatidyl ethanolamine(DOPE)/3beta[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterolDC-Cholin one to one mixtures (Gao et al., (1991) Biochim. Biophys. Acta 1065,8-14), spermine, spermidine, lipopolyamines (Behr et al., BioconjugateChem, 1994, 5: 382-389), lipophilic polylysines (LPLL) (Zhou et al.,(1991) Biochim. Biophys. Acta 939, 8-18),[[(1,1,3,3-tetramethylbutyl)cre-soxy]ethoxy]ethyl]dimethylbenzylammoniumhydroxide (DEBDA hydroxide) with excess phosphatidylcholine/cholesterol(Ballas et al., (1988) Biochim. Biophys. Acta 939, 8-18),cetyltrimethylammonium bromide (CTAB)/DOPE mixtures (Pinnaduwage et al,(1989) Biochim. Biophys. Acta 985, 33-37), lipophilic diester ofglutamic acid (TMAG) with DOPE, CTAB, DEBDA, didodecylammonium bromide(DDAB), and stearylamine in admixture with phosphatidylethanolamine(Rose et al., (1991) Biotechnique 10, 520-525), DDAB/DOPE (TransfectACE,GIBCO BRL), and oligogalactose bearing lipids. Exemplary transfectionenhancer agents that increase the efficiency of transfer include, forexample, DEAE-dextran, polybrene, lysosome-disruptive peptide (Ohmori NI et al, Biochem Biophys Res Commun Jun. 27, 1997; 235(3):726-9),chondroitan-based proteoglycans, sulfated proteoglycans,polyethylenimine, polylysine (Pollard H et al. J Biol Chem, 1998 273(13):7507-11), integrin-binding peptide CYGGRGDTP, linear dextrannonasaccharide, glycerol, cholesteryl groups tethered at the 3′-terminalinternucleoside link of an oligonucleotide (Letsinger, R. L. 1989 ProcNatl Acad Sci USA 86: (17):6553-6), lysophosphatide,lysophosphatidylcholine, lysophosphatidylethanolamine, and 1-oleoyllysophosphatidylcholine.

In some situations it may be desirable to deliver the nucleic acid withan agent that directs the nucleic acid-containing vector to targetcells. Such “targeting” molecules include antibodies specific for acell-surface membrane protein on the target cell, or a ligand for areceptor on the target cell. Where liposomes are employed, proteinswhich bind to a cell-surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake.Examples of such proteins include capsid proteins and fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. In other embodiments,receptor-mediated endocytosis can be used. Such methods are described,for example, in Wu et al., 1987 or Wagner et al., 1990. For review ofthe currently known gene marking and gene therapy protocols, seeAnderson 1992. See also WO 93/25673 and the references cited therein.For additional reviews of gene therapy technology, see Friedmann,Science, 244: 1275-1281 (1989); Anderson, Nature, supplement to vol.392, no 6679, pp. 25-30 (1998); Verma, Scientific American: 68-84(1990); and Miller, Nature, 357: 455460 (1992).

Methods of Determining Enzymatic Activity of LPLA2

As indicated above, the LPLA2 proteins of used herein have transacylaseactivity. Such an enzyme activity is readily determined using assaysknown to those of skill in the art. As the LPLA2 proteins is generallyspecific for PE and PC, the substrates in any transacylase enzyme assaycomprise, for example, one or both of these phospholipids. In oneaspect, in an exemplary general assay, liposomes comprisingdioleoylphosphatidylcholine (60.5 mol %), PE (27.3 mol %) and dicetylphosphate (12.3 mol %) are used as the acyl group donor for the enzymebeing tested. In exemplary assays, such liposomes are formed by mixingconstituent lipids in chloroform and drying the mixture under a streamof nitrogen. Fifty mM sodium citrate (pH 4.5) is added to the driedlipids at a volume of 1 ml/128 nmol of lipid phosphorus. The lipids arecaused to disperse into the buffer for 8 min in an ice-water bath usinga probe sonicator. This procedure creates donor liposomes for the enzymeassay. Those skilled in the art understand that similar liposomecommercially available.

Donor liposomes containing e.g., 64 nmol of phospholipid are incubatedwith 10 nmol of N-acetylsphingosine (NAS) or 5 nmol of [3H]NAS (10,000cpm), 5 μg of bovine serum albumin, and LPLA2 protein containingpreparation at 37° C. in a total volume of 500 μl of 40 mM sodiumcitrate (pH 4.5). The reaction is terminated by adding 3 ml ofchloroform/methanol (2:1) plus 0.3 ml of 0.9% (w/v) NaCl. Aftercentrifugation for 5 min at 800×g, the lower layer is transferred intoanother glass tube and dried down under a stream of nitrogen gas. Thelipid extract is then analyzed using e.g., high performance applied thinlayer chromatography (HPTLC) to confirm the presence of1-O-acyl-N-acetylsphingosine (1-O-acyl-NAS). In exemplary embodiments,the HPTLC plate and developed in a solvent system consisting ofchloroform/acetic acid (9:1). Of course the lipid catabolism also isreadily analyzed using other techniques, such as gas chromatography,HPLC and the like.

In an exemplary embodiment, an HPTLC assay is performed usingnonradioactive NAS, the TLC plate is dried, sprayed with 8% (w/v) CuSO4pentahydrate in water/methanol/concentrated H3PO4 (60:32:8), and charredfor 15 min at 150° C. An image of the plate is taken by a scanner (UMAXAstra Scanner 2200) connected to a computer and scanned by the NIH Imageprogram (Version 1.62) to estimate the density of each band. Knownamounts of ceramide are used to obtain a standard curve. In an exemplaryassay using radioactive NAS, 1-O-acyl-NAS is detected under a UV lightwith primulin spray, scraped, and counted. Other assays for enzymeactivity are known to those of skill in the art and are readily adaptedto determine whether a given LPLA2 variant or fragment possesses therequisite transacylase or phospholipase activity.

In addition to the above in vitro enzyme assays, those skilled in theart also test the enzymatic activity of any of the LPLA2 proteincompositions described herein by determining the presence of tingiblebody macrophages in a sample prior to and after contacting the samplewith the LPLA2 protein. Any of the above assays also are used to screenfor potential therapeutics that decrease intracellular levels oftingible body macrophages.

Methods of Screening for Modulators of LPLA2

The present invention also contemplates methods for identifyingadditional agents that decrease intracellular levels of tingible bodymacrophages through augmenting, increasing or otherwise stimulating theactivity of LPLA2, or increasing or otherwise stimulating the expressionof LPLA2.

In the screening assays of the present invention, the candidatesubstance may first be screened for basic biochemical activity—e.g., invitro stimulation of LPLA2 activity, and then tested for its ability toreduce accumulation of intracellular tingible body macrophages. To testthis effect, animal models exhibiting an accumulation of tingible bodymacrophages are known, e.g., LPLA2−/− knockout mouse model.

Other mouse models for macrophage defects known in the art are alsosuitable to test a candidate substance for its ability to reduceaccumulation of intracellular tingible body macrophages. These modelsinclude, but are not limited to: the Ro mouse model (Xue et al., ProcNatl Acad Sci USA 100: 7503-7508, 2003), the Tyro 3 mouse model (Lu etal., Science 293: 306-311, 2001), the c-mer mouse model (Cohen et al., JExp Med 196: 135-140, 2002), and the MFG-E8 mouse model (Hanayama etal., Science 304: 1147-1150, 2004).

a. Binding Assays

Preliminarily, candidate substances can be identified by screening formolecules that bind to LPLA2. Binding of a molecule to a target may, inand of itself, be stimulatory, due to steric, allosteric orcharge-charge interactions. In some aspects, this is performed insolution, in other aspects it is performed on a solid phase and isutilized as a first round screen to rapidly eliminate certain compoundsbefore moving into more sophisticated screening assays. In oneembodiment of this kind, the screening of compounds that bind to theLPLA2 of SEQ ID NO: 1 or a fragment thereof is provided.

The LPLA2 can be free in solution, fixed to a support, or expressed inor on the surface of a cell. Either the LPLA2 or the compound to bescreened is labeled, thereby permitting determining of binding. One maymeasure the amount of free label versus bound label to determinebinding.

A technique for high throughput screening of compounds is described inWO 94/03564. Large numbers of small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with, for example, LPLA2and washed. Bound polypeptide is detected by various methods.

LPLA2 can be fixed to a support by coating directly onto plates, or byusing non-neutralizing antibodies to immobilize the polypeptide to asolid phase. Alternatively, LPLA2 fusion proteins containing a reactiveregion (such as, for example, a terminal region) can be used to link theLPLA2 active region to a solid phase.

b. In Vitro Assays

Assays for determining the ability of a candidate substance to augment,increase or otherwise stimulate LPLA2 activity generally include thesteps of: i) contacting a LPLA2 of SEQ ID NOs: 1-288 with a candidatemodulator; ii) monitoring the activity of said LPLA2; and iii) comparingthe activity of LPLA2 in the presence and absence of said candidatesubstance; wherein an increase in the activity of said LPLA2 indicatesthat the candidate substance will augment, increase or otherwisestimulate LPLA2 activity. Exemplary assays for determining LPLA2activity are discussed above in the section entitled “Methods ofDetermining Enzymatic Activity of LPLA2.”

Significant changes in activity and/or expression include those that arerepresented by alterations in activity of at least about 30%-40%, and insome aspects, by changes of at least about 50%, with higher values ofcourse being possible.

Alternatively, the candidate substance is added to isolated peritonealmacrophages, e.g. macrophages from a animal model of lupus, and thenumber or phagocytic index of tingible body macrophages is determined inthe presence and absence of the added candidate substance.

c. In Vivo Assays

The present invention particularly contemplates the use of variousanimal models. As discussed above, there is a LPLA2−/− knockout mousemodel. Other animal models of lupus are also known in the art, forexample, the Ro mouse model, the Tyro 3 mouse model, the c-mer mousemodel, and the MFG-E8 mouse model.

Treatment of these animals with test compounds will involve theadministration of the compound, in an appropriate form, to the animal.Administration will be by any route that could be utilized for clinicalor non-clinical purposes, including but not limited to oral, nasal,buccal, or even topical. Alternatively, administration is byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are intravenous and other mechanisms fordelivery of the candidate substance locally to lymphatic tissue.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Such criteria include, but are notlimited to, clearance of apoptotic bodies, reduction in accumulation oftingible body macrophages, survival, reduction of SLE-associated signs,symptoms or complications, and improvement of general physical stateincluding activity. It also is possible to perform histologic studies ontissues from these mice, or to examine the molecular and morphologicalstate of the cells, which includes cell size, other morphologicalindicators or alteration in the expression of genes involved in SLEdisorders.

In the in vivo screening assays of the present invention, the compoundis administered to a model animal, over period of time and in variousdosages, and an alleviation of the accumulation of tingible bodymacrophages and associated lupus-like symptoms are monitored. Anyimprovement in one or more of these symptoms will be indicative of thecandidate substance being a useful modulator.

d. Screening for Increased Expression of LPLA2

Candidate substances are screened for their ability to increaseexpression of LPLA2 using techniques known in the art. For example,cells that normally express LPLA2, or cells that do not normally expressLPLA2 are contacted with a candidate substance for a period of time, andlevels of LPLA2 mRNA are monitored, e.g. using quantitative PCR methods.

e. Candidate Substances

As used herein the term “candidate substance” refers to any moleculethat may potentially act as a modulator of the LPLA2 of the presentinvention. In certain aspects, the candidate substance is a protein orfragment thereof, a small molecule inhibitor, or even a nucleic acidmolecule. In other aspects, the candidate substance is a non-specifictranscription factor, an upstream activator in a LPLA2 cascade, aninhibitor of a LPLA2 inhibitor, an inhibitor of a silencer (i.e., a DNAbinding protein that inhibits transcription) or gene therapy thatreplaces a natural LPLA2 promoter with a more active promoter. Rationaldrug design includes not only comparisons with known modulators ofphospholipases, but predictions relating to the structure of targetmolecules. Alternatively, rapid and efficient screening of entirelibraries of unrelated or related organic chemical compounds, orcombinatorially generated libraries (e.g., peptide libraries), ispossible. Combinatorial approaches also lend themselves to rapidevolution of potential drugs by the creation of second, third and fourthgeneration compounds related to the first.

Compounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples areassayed as candidates for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived or synthesized from chemicalcompositions or man-made compounds. Thus, it is understood that thecandidate substance identified by the present invention is apolypeptide, a polynucleotide, a small molecule inhibitor or any othercompound that is designed through rational drug design starting from aknown activator of a phospholipase A2 activity.

In some aspects of the invention, the candidate substance is a variantof LPLA2 prepared as described above. Such a variant is readily testedfor increased LPLA2 activity using any of the assays described herein.In other aspects of the invention, the candidate substance is ananti-idiotypic antibodies of LPLA2. Anti-idiotypes are generated byproducing antibodies specific for a given protein and then using such anantibody as an antigen to produce the anti-idiotypic antibody. As amirror image of a mirror image, the binding site of anti-idiotype is ananalog of the original antigen. Methods of making antibodies to antigenssuch as LPLA2 are known to those of ordinary skill in the art, and aredisclosed, e.g., in Sambrook et al. (2000) and Harlow and Lane (1988).Anti-idiotypic antibodies can be generated by methods known in the art,including, e.g., Greenspan et al., FASEB. J., 7:437 (1993).

Diagnostic Methods

The invention also provides methods for diagnosing systemic lupuserythematosus, determining susceptibility to systemic lupuserythematosus, and monitoring progression of systemic lupuserythematosus. Such methods involve assaying body fluid, cells, ortissue samples from patients for the presence or absence of LPLA2autoantibodies or for reduced or normal LPLA2 expression or activity,wherein the presence of LPLA2 autoantibodies suggests diagnosis of orsusceptibility to disease, and wherein a reduced LPLA2 expression oractivity suggests diagnosis of or susceptibility to disease.

Examples of body fluid samples include blood, serum, plasma, pleuralfluid, pulmonary or bronchial lavage fluid, synovial fluid, peritonealfluid, bone marrow aspirate, lymph, cerebrospinal fluid, ascites fluid,amniotic fluid, sputum, bladder washes, semen, urine, saliva, and tears.Examples of cells include white blood cells, B- or T-lymphocytes,peritoneal lymphocytes, macrophages or dendritic cells. Examples oftissues include biopsies from spleen, kidney, lung, lymph nodes or bloodvessels. Suitable assay methods are well known in the art andillustrated in the examples herein.

In one aspect, such methods comprise determining lysosomal phospholipaseA2 (LPLA2) enzymatic activity in a sample from an individual. Theenzymatic activity levels of the patient can be compared to those of anormal individual, or alternatively can be compared to previous LPLA2enzymatic activity levels from the same individual. Detection of LPLA2enzymatic activity that is decreased in the individual compared to LPLA2enzymatic activity in a normal individual (wherein said normalindividual is known not to suffer from SLE), is suggestive of thediagnosis of SLE or systemic lupus erythematosus, and/or susceptibilityto SLE or systemic lupus erythematosus. Detection of LPLA2 enzymaticactivity that is decreased in the individual compared to a priordetermination of LPLA2 enzymatic activity in the same individual is alsosuggestive of the diagnosis of SLE or systemic lupus erythematosusand/or susceptibility to SLE or systemic lupus erythematosus.

In another aspect, such methods comprise determining LPLA2 expression incells or tissue from an individual. The LPLA2 expression levels of thepatient can be compared to those of a normal individual, oralternatively can be compared to previous LPLA2 expression levels fromthe same individual. Detection of LPLA2 expression level that isdecreased in the individual compared to LPLA2 expression level in anormal individual (wherein said normal individual is known not to sufferfrom SLE), is suggestive of the diagnosis of SLE or systemic lupuserythematosus and/or susceptibility to SLE or systemic lupuserythematosus. Detection of LPLA2 expression level that is decreased inthe individual compared to a prior LPLA2 expression level in the sameindividual is also suggestive of the diagnosis of SLE or systemic lupuserythematosus and/or susceptibility to SLE or systemic lupuserythematosus.

In yet another aspect, such methods comprise detecting lysosomalphospholipase A2 (LPLA2) autoantibodies in a sample from an individual.Detecting increased LPLA2 autoantibodies in the individual compared toLPLA2 autoantibodies in a normal individual (wherein said normalindividual is known not to suffer from SLE) is suggestive of thediagnosis of SLE or systemic lupus erythematosus and/or susceptibilityto SLE or systemic lupus erythematosus. Detecting increased LPLA2autoantibodies in a sample from an individual compared to prior levelsof LPLA2 autoantibodies in the same individual is suggestive of thediagnosis of SLE or systemic lupus erythematosus and/or susceptibilityto SLE or systemic lupus erythematosus.

In further aspects, the invention provides mehods of monitoringprogression of SLE or systemic lupus erythematosus or rheumatoidarthritis in an individual. Such methods comprise periodic determinationof levels of LPLA2 enzymatic activity, or levels of LPLA2 expression, orlevels of LPLA2 autoantibodies, in samples taken from the individualover time. Levels can be measured every week, two weeks, three weeks, ormonthly, or every 2, 3, 4, 5, 6, or 12 months. Decreasing levels ofLPLA2 enzymatic activity over time, or decreasing levels of LPLA2expression over time, or increasing levels of LPLA2 autoantibodies overtime are suggestive of progression of systemic lupus erythematosus.

In related aspects, the invention provides methods of monitoringeffectiveness of drug therapy of SLE or systemic lupus erythematosus orrheumatoid arthritis. In some embodiments, the methods involvemonitoring levels of exogenously administered LPLA2 enzyme, or fragment,variant or derivative thereof. In other embodiments, such methodscomprise periodic determination of levels of LPLA2 enzymatic activity,or levels of LPLA2 expression, or levels of LPLA2 autoantibodies, insamples taken from the individual over time. Levels can be measuredevery week, two weeks, three weeks, or monthly, or every 2, 3, 4, 5, 6,or 12 months. Increasing levels of LPLA2 enzymatic activity over time,or increasing levels of LPLA2 expression over time, or decreasing levelsof LPLA2 autoantibodies over time are suggestive of therapeuticefficacy.

Alternatively, such methods involve determining the presence or absenceof a polymorphism in the LPLA2 gene that results in reduced expressionor activity, wherein the presence of such a polymorphism suggestsdiagnosis of or susceptibility to disease. Such methods may optionallyinvolve amplifying the DNA or RNA from the sample. Such methods may becarried out by any means known in the art, including (a) hybridizing theDNA or RNA with an oligonucleotide probe and detecting hybridization;(b) electrophoretic analysis; (c) restriction fragment lengthpolymorphism analysis; and (d) nucleotide sequence analysis. Forhybridization, the probe is contacted with DNA from the sample underconditions that are sufficient to allow specific hybridization of thenucleic acid probe. “Specific hybridization”, as used herein, indicatesexact hybridization (e.g., with no mismatches). Specific hybridizationcan be performed under high stringency conditions or moderate stringencyconditions, preferably high stringency. See Current Protocols inMolecular Biology, Ausubel, F. et al., eds., John Wiley & Sons.Alternatively, the probe is contacted with RNA from the sample underspecific hybridization conditions, to identify the presence of apolymorphism or a particular splicing variant. Mutation analysis byrestriction digestion can be used to detect a polymorphism(s), if themutation or polymorphism in the nucleic acid results in the creation orelimination of a restriction site. DNA, possibly including flankingsequences, is amplified and digested with a restriction enzyme thatcleaves at the restriction site that has been created or eliminated bythe polymorphism. RFLP analysis is conducted as known in the art (seeCurrent Protocols in Molecular Biology, supra). The digestion pattern ofthe relevant DNA fragment indicates the presence or absence of thepolymorphism. Sequencing of the relevant DNA or RNA, and comparison withnormal gene sequence, can also be used to detect polymorphisms Dot-blothybridization of amplified DNA or RNA with allele-specificoligonucleotide (ASO) probes (see, for example, Saiki, R. et al., Nature324:163-166 (1986)) is also known in the art. An “allele-specificoligonucleotide” (also referred to herein as an “allele-specificoligonucleotide probe”) is an oligonucleotide of approximately 10-50base pairs, for example, approximately 15-30 base pairs, thatspecifically hybridizes to a gene containing a polymorphism associatedwith disease.

Methods of Treating Autoimmune Disorders

As described herein throughout, it has been discovered that LPLA2proteins are used to improve the clearance of apoptotic bodies and/orreduce accumulation of intracellular tingible body macrophages. As such,the invention provides any LPLA2 of SEQ ID NO: 1-288, or a fragment,variant or derivative that retains the desired biological activity foruse in the treatment of any disorder displaying the symptom ofaccumulation of tingible body macrophages. In a related aspect, theinvention provides methods of treatment using agents that decreaseintracellular levels of tingible body macrophages through augmenting,increasing or otherwise stimulating the activity of LPLA2, or increasingor otherwise stimulating the expression of LPLA2.

In certain aspects, the methods of the invention are useful in thetreatment of SLE, including systemic lupus erythematosus, drug-inducedlupus, neonatal lupus, and cutaneous lupus. However, it should beunderstood that in one aspect, the methods of the invention are usefulin the treatment of any and all disorders that manifest in theaccumulation of intracellular tingible body macrophages, including otherautoimmune disorders such as rheumatoid arthritis. The invention alsospecifically contemplates use of any of the therapeutic agents describedherein in the preparation of a medicament for the treatment of theabove-described disorders.

To achieve the appropriate therapeutic outcome, either by administrationof the LPLA2-related compositions alone or in combination with othertherapeutic modalities, one generally administers to the subject thetherapeutic protein composition in an effective amount. “Effectiveamount” is an amount effective to produce the desired therapeuticoutcome reproducibly, i.e., an alleviation of one or more of the signsor symptoms or complications of the disease.

Administration of these compositions according to the present inventionwill be via any route so long as the target tissue is available via thatroute. However, other conventional routes of administration, e.g.,parenterally, subcutaneous, intravenous, intradermal, intramuscular,intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar,intrapulmonary, intratracheal instillation, bronchial instillation,aerosol, sublingual, oral, nasal, anal, vaginal, or transdermaldelivery, or by surgical implantation at a particular site. Specificallycontemplated are intravenous and other mechanisms for delivery of thecandidate substance locally to lymphatic tissue.

The treatment may consist of a single dose or a plurality of doses overa period of time. Administration of the compositions can be systemic orlocal, and may comprise a single site injection or infusion of atherapeutically-effective amount of the LPLA2 protein composition.Alternatively, it is contemplated that the therapeutic composition maybe delivered to the patient at multiple sites. The multipleadministrations may be rendered simultaneously or may be administeredover a period of time. Additional therapy may be administered on aperiod basis, for example, daily, weekly, or monthly.

In certain embodiment, parenteral administration of the therapeuticcompounds is carried out with an initial bolus followed by continuousinfusion to maintain therapeutic circulating levels of drug product.Those of ordinary skill in the art will readily optimize effectivedosages and administration regimens as determined by good medicalpractice and the clinical condition of the individual patient.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agents and the routes of administration. Depending on the route ofadministration, a suitable dose is calculated according to body weight,body surface areas or organ size. The availability of animal models isparticularly useful in facilitating a determination of appropriatedosages of a given therapeutic. In one aspect, the dosage is determinedusing an animal model, such as the LPLA2−/− knockout mouse modeldescribed herein, and modified and adapted to use in higher mammals.

Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation. The final dosage regimen willbe determined by the attending physician, considering factors whichmodify the action of drugs, e.g., the drug's specific activity, severityof the damage and the responsiveness of the patient, the age, condition,body weight, sex and diet of the patient, the severity of any infection,time of administration, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired, and other clinicalfactors. As studies are conducted, further information will emergeregarding appropriate dosage levels and duration of treatment forspecific diseases and conditions.

In certain embodiments, the compositions are administered alone, inother embodiments the compositions are administered in conjunction withother therapeutics directed to the disease or directed to other symptomsthereof. Dosage levels of the order from about 0.01 mg to 30 mg perkilogram of body weight per day, for example from about 0.1 mg to 10mg/kg.

It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention are useful in fields of humanmedicine and veterinary medicine. Thus the subject to be treated is amammal, such as a human or other mammalian animal. For veterinarypurposes, subjects include for example, farm animals including cows,sheep, pigs, horses and goats, companion animals such as dogs and cats,exotic and/or zoo animals, laboratory animals including mice rats,rabbits, guinea pigs and hamsters; and poultry such as chickens, turkeyducks and geese. The patient being treated is of any age, for example,between the ages of 10-50 years, age 20 or less, or age 10 or less.

In addition, it is contemplated that the peptide/protein-basedcompositions of the present invention are used in combination with anypresent treatments for disorders associated with an abnormalaccumulation of tingible body macrophages. For example, in certainembodiments, it is contemplated that the methods of the invention areuseful in combination with known SLE therapy. Compositions comprisingLPLA2, or a fragment, variant or derivative thereof, are administeredbefore, after or during such therapy.

Combination therapy with other agents or drugs for treating SLE include,but are not limited to, anti-inflammatory drugs (NSAIDs) such asaspirin, ibuprofen (Motrin), naproxen (Naprosyn) and sulindac(Clinoril), cyclooxygenase-2 (COX-2) inhibitors including Celecoxib(Celebrex), rofecoxib (Vioxx), valdecoxib (Bextra) and Meloxicam(Mobicox), corticosteroids such as prednisone, hydrocortisone,methylprednisolone, and dexamethasone, hydroxychlooquine (Plaquenil),mycophenolate mofetil (Cellcept), chloroquine (Aralen), quinacrine,rituximab (Rituxan), dapsone, retinoic acid (Retin-A), plasmapheresis,cytotoxic drugs such as methotrexate (Rheumatrex, Trexall), azathioprine(Imuran), cyclophosphamide (Cytoxan), chlorambucil (Leukeran), andcyclosporine (Sandimmune) and the costimulation blocker CTLA41G. Thecombined therapies contemplated herein, i.e., combinations ofLPLA2-based compositions with SLE drugs are, in one aspect, administeredin a combined amount effective to produce a decrease in the accumulationof tingible body macrophages. Such a combined administration in someaspects alleviates one or more signs, symptoms or complications that areassociated with an accumulation of intracellular tingible bodymacrophages.

Pharmaceutical Compositions

Pharmaceutical compositions for administration according to the presentinvention can comprise at least one LPLA2-derived protein (e.g., aprotein of SEQ ID NOs: 1-288, a variant or derivative thereof or anyother LPLA2-derived protein that stimulates the catabolism of tingiblebody macrophages). The pharmaceutical compositions also include anotheragent that is used for the treatment of SLE, e.g., NSAIDs,corticosteroids or cytotoxic agents. Each of these preparations is insome aspects provided in a pharmaceutically acceptable form optionallycombined with a pharmaceutically acceptable carrier. These compositionsare preferably sterile and may be administered by any methods thatachieve their intended purposes.

The instant compositions can be formulated for various routes ofadministration, for example, by oral administration, by transmucosaladministration (including pulmonary and nasal administration),parenteral administration (including subcutaneous administration),transdermal (topical) administration or by rectal administration, aswell as intrathecal, intravenous, intramuscular, intraperitoneal,intranasal, intraocular or intraventricular injection. The compound orcompounds of the instant invention can also be administered in a localrather than a systemic fashion, such as injection as a sustained releaseformulation.

The optimal pharmaceutical formulation will be determined by one ofskill in the art depending on the route of administration and thedesired dosage. Such formulations may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theadministered agents.

Besides those representative dosage forms described herein,pharmaceutically acceptable excipients and carriers are generally knownto those skilled in the art and are thus included in the instantinvention. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey

Injectable dosage forms for parenteral administration generally includeaqueous suspensions or oil suspensions, which may be prepared using asuitable dispersant or wetting agent and a suspending agent. Generally,this will entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals. Injectable forms may be in solution phase or provided as alyophilized powder suitable for reconstitution as a solution. Both areprepared with a solvent or diluent. Acceptable solvents or vehiclesinclude sterilized water, Ringer's solution, or an isotonic aqueoussaline solution. Alternatively, sterile oils may be employed as solventsor suspending agents. Typically, the oil or fatty acid is non-volatile,including natural or synthetic oils, fatty acids, mono-, di- ortri-glycerides. For injection, the formulations may optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations of these. The compounds may be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion. A unit dosage form for injection may be in ampoules or inmulti-dose containers.

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, and sustained-releasing as described below.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release. The instant compositions mayalso comprise, for example, micelles or liposomes, or some otherencapsulated form, or may be administered in an extended release form toprovide a prolonged storage and/or delivery effect. Therefore, thepharmaceutical formulations may be compressed into pellets or cylindersand implanted intramuscularly or subcutaneously as depot injections oras implants such as stents. Such implants may employ known inertmaterials such as silicones and biodegradable polymers.

Any of the above dosage forms containing effective amounts are wellwithin the bounds of routine experimentation and therefore, well withinthe scope of the instant invention.

Compositions within the scope of this invention include all compositionscomprising at least one LPLA2-derived protein according to the presentinvention in an effective amount. In some aspects, such treatment willresult in an alleviation of one or more signs or symptoms orcomplications of SLE discussed above.

One will generally desire to employ appropriate salts and buffers torender the compositions stable and allow for uptake of the compositionsat the target site. Generally the protein compositions of the inventionare provided in lyophilized form to be reconstituted prior toadministration. Buffers and solutions for the reconstitution of thetherapeutic agents may be provided along with the pharmaceuticalformulation to produce aqueous compositions of the present invention foradministration. Such aqueous compositions will comprise an effectiveamount of each of the therapeutic agents being used, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.The phrase “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce adverse,allergic, or other untoward reactions when administered to an animal ora human. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the therapeutic compositions, its use intherapeutic compositions is contemplated. Supplementary activeingredients also are incorporated into the compositions.

Methods of formulating proteins and peptides for therapeuticadministration also are known to those of skill in the art. In certainembodiments, the active compounds are prepared for administration assolutions of free base or pharmacologically acceptable salts in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions also are prepared in glycerol, liquid polyethylene glycols,and mixtures thereof and in oils. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms.

The pharmaceutical forms suitable for injection include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In all casesthe form must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. In some aspects,the carrier is a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity is maintained, for example, bythe use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms is broughtabout by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions is brought about by the use in the compositionsof agents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, the methodsof preparation are vacuum-drying and freeze-drying techniques whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

In some aspects, the compositions of the present invention areformulated in a neutral or salt form. Pharmaceutically-acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups also are derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution is suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration.

“Unit dose” is defined as a discrete amount of a therapeutic compositiondispersed in a suitable carrier.

The present invention also contemplates kits for use in the treatment ofthe intracellular accumulation of tingible body macrophages, e.g. SLE orsystemic lupus erythematosus. Such kits include at least a first sterilecomposition comprising the proteins/peptides described above in apharmaceutically acceptable carrier. Another component is optionally asecond therapeutic agent for the treatment of the disorder along withsuitable container and vehicles for administrations of the therapeuticcompositions. The kits may additionally comprise solutions or buffersfor suspending, diluting or effecting the delivery of the first andsecond compositions. The kits may further comprise catheters, syringesor other delivering devices for the delivery of one or more of thecompositions used in the methods of the invention. The kits may furthercomprise instructions containing administration protocols for thetherapeutic regimens.

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Characterization of LPLA2−/− Knockout Mice

Double conditional gene targeting was employed to elucidate the functionof LPLA2. LPLA2-deficient mice (LPLA2−/−) were generated by the systemicdeletion of exon 5 of the LPLA2 gene, which encodes the lipase motifessential for the phospholipase A2 activity (Hiraoka, M., Abe, A., Lu,Y., Yang, K., Han, X., Gross, R. W., and Shayman, J. A. Mol Cell Biol26, 6139-6148 2006). The survival of the LPLA2−/− mice was normal.LPLA2−/− mouse mating pairs yielded normal litter sizes, indicating thatthe gene deficiency did not impair fertility or fecundity. Alveolarmacrophages from wild-type but not LPLA2−/− mice readily degradedradiolabeled phosphatidylcholine. By three months of age, a markedaccumulation of phospholipids, in particular phosphatidylethanolamineand phosphatidylcholine, was found in the alveolar macrophages, theperitoneal macrophages and the spleens of LPLA2−/− mice. Theultrastructural examination of LPLA2−/− mouse alveolar and peritonealmacrophages revealed the appearance of foam cells with lamellarinclusion bodies, a hallmark of cellular phospholipidosis. This findingwas not surprising since very high expression of LPLA2 mRNA and proteinhad been observed in alveolar macrophages, consistent with a role forLPLA2 in surfactant catabolism (Abe, A., Hiraoka, M., Wild, S.,Wilcoxen, S. E., Paine, R., 3rd, and Shayman, J. A. J Biol Chem 279,42605-42611 2004).

By six months of age the LPLA2 null mice began to develop signs oflymphoproliferation. Splenic enlargement was readily apparent as wasadenopathy. By 18 months of age the spleens were greater than 10 timesthe weight of those obtained from the wild type mice and diffuselymphadenopthy was readily apparent. Female mice demonstrated asignificantly greater predilection toward increased organ weight thanmale mice. By 20 months of age the knockout mice displayed weight losswith signs of wasting as evidenced by loss of subcutaneous fat (FIG. 2).Histological analysis of the lymphoid organs revealed the presence ofmacrophages with a foam cell phenotype and the loss of stromalarchitecture (FIG. 3). Flow cytometric analysis of the spleniclymphocytes revealed a polyclonal expansion of both B and T cells.

Histological analysis of the kidneys of LPLA2−/− mice revealed thepresence of an immune complex glomerulonephritis with a “full house”pattern of deposition. LPLA2−/− mice showed proliferative changes andloss of capillary loop patency, consistent with a proliferativeglomerulonephritis. Corresponding transmission electron micrographsdemonstrated foot process effacement, immune complex deposition, and thepresence of electron dense apoptotic bodies. Immunofluorescencedemonstrated increased Ig and Clq deposition in the glomeruli of thenull (LPLA2−/−) verse wild type (LPLA2 +/+) mice (FIG. 4A). Bytransmission electron microscopy, immune complex deposition was evidentin various glomerular compartments including mesangial, subepithelial,and subendothelial regions. Serum immunoglobulin levels were markedlyelevated in the older mice and anti-dsDNA titers were significantlyelevated in an age dependent fashion (FIG. 4B) as was the presence ofANAs (FIG. 4C). The spleens were characterized by numerous enlargedmacrophages and the presence of massive amounts of tingible bodiesconsistent with the inability to clear apoptotic cells (FIG. 4D). Frozensections of KO LPLA2−/− mice spleens were stained for CD68 (red) andapoptotic bodes with a TUNEL stain (green) (FIG. 4D).

A primary mode of action of LPLA2 is the degradation of extracellularphospholipids to form free fatty acids and lyso-phosphatidylcholine andlyso-phospharidylethanomine. LPLA2 is secreted from macrophagesfollowing activation by ligand, subsequently binds to mannose receptorson the macrophage and is trafficed back to the lysosome. To assessuptake and co-localization of LPLA2 and lysosomes, macrophages fromLPLA2−/− KO mice were treated with recombinant LPLA2, fixed and stainedwith an anti-LPLA2 monoclonal antibody (green) (FIG. 5A) or an antibodyagainst the lysosomal marker LAMP-1 (red) (FIG. 5B). FIG. 5C shows anoverlay of FIGS. 5A and 5B where yellow indicates co-localization ofLPLA2 and the lysosome.

Additional changes in the 18 month and older mice included thedevelopment of renal insufficiency and proteinuria. FIG. 6 shows that 24hour protein excretion is more than 2-fold elevated in the LPLA2−/− micecompared to wild type mice. Creatinine concentrations were also elevatedin LPLA2−/− mice (0.63+/−0.22) versus the LPLA2+/+ mice (0.25+/−0.06).

Example 2 Impaired Digestion of Apoptotic Bodies can be Recapitulated inPeritoneal Macrophages from LPLA2−/− Mice

In order to determine whether the appearance of apoptotic bodies withinthe spleens and glomeruli of LPLA2−/− knockout mice resulted from ageneralized defect in the digestion of endocytosed apoptotic cells, aLPLA2 defect was recapitulated in vitro.

Peritoneal macrophages were obtained following intraperitonealthioglycolate injection of 1 year old wild type and null mice.Thymocytes were harvested and apoptosis induced by 24 hour treatmentwith dexamethasone. LPLA2 +/+ (WT) and LPLA2−/− (KO) macrophages wereincubated with wild type apoptotic thymocytes for 0.5, 1, 2, and 4hours. After incubation, the plates were washed and the residualapoptotic bodies were measured by co-staining of the macrophages withanti-CD68 and TUNEL (FIG. 7A-7H). No differences were observed in therate of uptake or of the peak number of apoptotic bodies incorporatedbetween the wild type and null macrophages. However, a marked differencewas observed in the persistence of apoptotic bodies in the LPLA2−/−macrophages at 4 hours (FIG. 7H). When expressed as a phagocytic index,a highly significant difference was observed between the wild type andnull macrophages (FIG. 8).

B and T cell stimulation assays were used to ascertain whetherdifferences exist in the responsiveness between LPLA2−/− and LPLA2 +/+mice. B and T cells were isolated from the spleens of 8 month old femalemice and stimulated with antibodies to CD40 (FIG. 9A), CD3 (FIG. 9B),and CD3 and CD28 (FIG. 9C) in order to measure B cell stimulation, Tcell stimulation, or T cell co-stimulation respectively. Cellproliferation was measured by tritiated thymidine incorporation 3 daysfollowing isolation and stimulation. No difference was observed in Bcell responsiveness to CD40. However, the LPLA2−/− T cells were lessresponsive to both direct stimulation with CD3 and co-stimulation withCD3 and CD28 (FIGS. 9B and 9C). This latter response is opposite to thatreported in the G2A knockout mouse. Mixed lymphocyte reaction (MLR)assays were also performed using autologous, syngeneic, and allogeneicstimulators (FIG. 9D). The LPLA2−/− response to allogeneic stimulationwas significantly less than that observed in the wild type T cells. Thismay be significant in that several studies have been reporteddemonstrating a decrease in MLR activity in lupus patients (Kuntz etal., J Clin Invest. 63(1):151-153, 1979).

Example 3 LPLA2 is Secreted from Macrophages Following Activation and isReincorporated into Macrophages Via a Mannose Receptor

LPLA2 has been reported to be secreted from macrophages followingactivation by ligand (Abe et al., J Immunol 181: 7873-7881, 2008). Forexample, the binding and phagocytosis of zymosan result in the timedependent release of LPLA2 from alveolar macrophages.

The ability of macrophages from LPLA2−/− mice to recognize, endocytose,and traffic recombinant LPLA2 was studied as follows. LPLA2 nullalveolar macrophages were exposed to His-tagged mouse LPLA2 for 1 day atconcentrations varying between 0.3 and 10 μg/ml. Cell incorporation wasmeasured by Western blotting and confocal microscopy using eitheranti-His antibody or a monoclonal antibody raised to LPLA2. Aconcentration dependent increase in cellular LPLA2 was easily detectedand confirmed by enzyme activity measurements. When treated cells werestained with both anti-LPLA2 and Lamp-1 antibodies, co-localization ofthe recombinant enzyme and the lysosome protein were readily detected.

To ascertain whether the recognition and incorporation of therecombinant LPLA2 was through recognition by a mannose versusmannose-phosphate receptor, the incorporation experiments were repeatedin the presence of either 10 mM α-methyl-mannoside or 10 mMmannose-6-phosphate. α-Methyl-mannoside but not mannose-6-phosphateblocked the incorporation of LPLA2 as measured by cellular1-O-acyl-ceramide synthase activity, immunoblotting, and confocalmicroscopy. Finally, the ability of the recombinant enzyme to “rescue”the cellular phenotype was confirmed by measuring the cellularphospholipid content. A concentration dependent decrease in cellphosphatidylcholine and phosphatidylethanolamine was observed.Catalytically inactive enzyme did not lower the phospholipid levels.

Collectively, these data confirm that LPLA2 is secreted by macrophagesin response to zymosan and once secreted can be reclaimed through amannose receptor dependent process. These data also raise thepossibility that a primary mode of action of LPLA2 may be thedegradation of extracellular phospholipids to form free fatty acids andlyso-PC and lyso-PE.

Example 4 Cationic Amphiphilic Drugs Inhibit LPLA2 Activity and Resultin Cellular Phospholipidosis

Ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol(PDMP) has been observed to inhibit acidic transacylation of ceramidelocalized to the lysosomal fraction of cells. PDMP is structurallysimilar to drugs that are chemically characterized as cationicamphiphilic drugs. The possibility that such cationic amphiphilic drugsmight cause cellular phospholipidosis through a comparable mechanism wastested as follows.

MDCK cells were treated for 1 or seven days with the cationicamphiphilic drugs PDMP, amiodarone, and tetracycline. LPLA2 activity wasmeasured as the transacylation of N-acetylsphingosine. Amiodarone is aprototypic agent that causes phospholipidosis (and SLE); tetracyclinehas no known association with either lupus or phospholipidosis.Amiodarone and PDMP were both associated with a concentration dependentinhibition of LPLA2 activity as measured by ceramide transacylation(FIG. 10A). Amiodarone and PDMP but not tetracycline displayedcomparable IC50s for enzyme inhibition. Tetracycline was withoutinhibitory effect. Amiodarone treatment also resulted in a timedependent increase in cell phospholipids, most notably PC and PE. Themajor cellular phospholipids were assayed following 1 or 7 days ofexposure to the CAD. Highly significant changes in the LPLA2 substratesPE and PC were observed by 7 days of treatment but no changes insphingomyelin (SM) were observed (FIG. 10B).

The inhibition of LPLA2 activity by amiodarone and other cationicamphiphiles could have resulted from the direct binding of these drugsto the enzyme through a competitive or non-competitive effect. Analternative mechanism was considered, the disruption of theelectrostatic interaction between LPLA2 and the anionic lysosomalmembrane. Lysosomal membranes are characterized by the presence oflyso-bis-phosphatidic acid, an anionic phospholipid found exclusively inacidic cellular organelles most notably the late endosome and lysosome.

To address this possibility, the 1-O-acylceramide synthase activity ofLPLA2 was studied in liposomes in which the anionic lipid content wasvaried by the addition of sulfatide, a negatively charged, sulfatecontaining glycolipid that is not a substrate for LPLA2. Di-octanoyl-PC(DOPC) was used as the liposomal substrate. The reaction mixturecontained 48 mM sodium citrate (pH 4.5), 10 μg/ml BSA, liposomes (127 μMphospholipid) and 14.5 ng/ml of recombinant mouse LPLA2 in 500 μl oftotal volume. Liposomes consisting of DOPC/NAS (3:1, molar ratio) orDOP/sulfatide/NAS (3:0.3:1, molar ratio) were incubated with the enzymefor 5, 10, 15, 30 and 45 min at 37° C. The reaction products wereextracted and separated by an HPTLC plate using a solvent systemconsisting of chloroform/acetic acid (9:1, v/v). The reaction product,1-O-oleoyl-NAS, quantified by scanning the plate, was plotted againsttime (FIG. 11A). Liposomes consisting of DOPC/galactosylceramide/NAS orDOPC/sulfatide/NAS with a different molar ratio were incubated with therecombinant enzyme at 37° C. (FIG. 11B). A time dependent increase inthe transacylation activity was observed in the presence of sulfatide(FIGS. 11A and 11B) which increased as a function of the molar ratio ofsulfatide.

Example 5 Cationic Amphiphilic Drugs Interfere with the ElectrostaticInteractions Between LPLA2 and Anionic Lipid Membranes

The effects of ionic strength, pH and amiodarone on LPLA2 enzymaticactivity and on the electrostatic interaction between LPLA2 andliposomes were determined.

The reaction mixture contained 0-500 mM NaCl, 48 mM sodium citrate (pH4.5), 10 μg/ml BSA, liposomes (127 μM phospholipid) and either 14.5ng/ml of recombinant mouse LPLA2 (+) or 7.8 μg protein/ml of the solublefraction obtained from MDCK cells transfected with mouse LPLA2 (+) in500 μl of total volume. Before starting the reaction, liposomesconsisting of DOPC, sulfatide and NAS (3:0.3:1, molar ratio) werepre-incubated for 5 min at 37° C. The reaction was initiated by addingthe recombinant LPLA2 and carried out at 37° C. The reaction productswere extracted and separated by an HPTLC plate using a solvent systemconsisting of chloroform/acetic acid (9:1, v/v). One of the products,1-O-oleoyl-NAS, quantified by scanning the plate, was plotted againstNaCl concentration (FIG. 12A). Liposomes consisting of DOPC sulfatideand NAS were pre-incubated with a different concentration of cationicamphiphilic drug, amiodarone, for 5 mM at 37° C. and then incubated withthe recombinant LPLA2 (FIG. 12C). The reaction was carried out in sodiumcitrate/sodium phosphate buffer with various pH (FIG. 12B).

As one would predict for an enzyme-liposome interaction that wasdependent on electrostatic interactions, increasing either the NaClconcentration or pH of the reaction mixture significantly inhibited thetransacylase activity of the enzyme (FIGS. 12A and 12B). Importantly, ifthe LPLA2 activity was simply measured as an esterase using the watersoluble substrate p-nitro-phenylbutyrate (ρ-NPB) in the absence ofliposomes, the pH dependence was largely lost. Increasing theconcentration of amiodarone, a classic cationic amphiphilic drug,similarly inhibited the enzyme activity (FIG. 12C).

In order to determine whether the disruption of the electrostaticinteraction between LPLA2 and the liposome could be measured as aphysical interaction, co-sedimentation of the enzyme and liposomes wasdetected after high speed ultracentrifugation and separation by SDS-PAGEwhile the ionic strength, pH, and presence of amiodarone were varied.Liposomes consisting of DOPC or DOPC/sulfatide (10:1, molar ratio) wereincubated with varying amounts of recombinant mouse LPLA2 in 500 μl of48 mM Na-citrate (pH 4.5) for 30 min on ice (FIG. 13A). Liposomesconsisting of DOPC, DOPC/sulfatide (10:1, molar ratio) orDOPC/galactosylceramide (10:1, molar ratio) were incubated with 4 μg ofthe recombinant LPLA2 or 16 μg of the recombinant LPLA2 with or without500 mM NaCl or 33 μM amiodarone (FIGS. 13B and 13C). The reactionmixtures were centrifuged for 1 h at 150,000 g at 4° C. Theconcentration of DOPC in these assays was 127 μM. The resultantprecipitates were briefly washed with cold 50 mM Na-citrate (pH 4.5) anddissolved with 40 μl of SDS-polyacrylamide gel electrophoresis samplingbuffer. The samples were separated by using 12% SDS-polyacrylamide gel.After electrophoresis, LPLA2 was detected by staining with CBB.

The physical association between LPLA2 and liposomes was increased inthe presence of sulfatide (FIG. 13B) and was inhibited with higher pH,NaCl concentration, or the presence of amiodarone (FIG. 13C). These dataindicate that cationic amphiphilic drugs inhibit LPLA2 activity byinterfering with the electrostatic interactions between LPLA2 andanionic lipid membranes.

Example 6 LPLA2 Gene Expression is Decreased in the Glomeruli of LupusPatients Compared to Patients with Other Glomerular Diseases

To determine whether the expression of LPLA2 differed between patientswith lupus and patients with other forms of glomerular disease, geneexpression profiles from patients were analyzed. The Division ofNephrology at the University of Michigan contains the Applied SystemsBiology Core Laboratory. This group has biobanked and analyzed the geneexpression profiles of renal biopsies from patients with a variety ofrenal disorders obtained from centers in Europe and the United States.Control biopsy specimens are obtained from healthy kidney donors at thetime of transplantation. At the time of biopsy, the glomerular andtubulointerstial compartments are separated by microdissection and themRNA profiles obtained. Analysis using statistical analysis bymicroarray (SAM) revealed a significantly lower expression level inlupus glomeruli compared to controls or biopsies from patients withdiabetes, IgA nephropathy, minimal change disease, or hypertensivenephrosclerosis (Table 2).

Fold Change SAM q-value SLE (glom) 0.857 0.009 n = 32 SLE (ti) n = 321.023 0.198 IgA (glom) n = 27 1.086 0.293 IgA (ti) n = 25 1.236 0.087MCD (glom) n = 6 1.142 0.336 MCD (ti) n = 5 1.012 NS Early DM (glom)1.329 0.001 n = 22 Early DM (ti) 0.970 0.242 n = 22 Prog DM (glom) 0.1160.552 n = 7 Prog DM (ti) n = 7 1.040 0.526 HTN (glom) 1.198 0.058 n = 15HTN (ti) n = 20 1.191 0.407

mRNA expression profiles were obtained from five different glomerulardiseases including early and progressive diabetic nephropathy (DM), IgAnephropathy, minimal change disease (MCD) and hypertensivenephrosclerosis (HTN). mRNA was obtained from renal biopsiesrepresenting a 5 mm core cortical sample from patients with a range ofestimated GFRs and in whom the diagnosis was established by pathologicalinvestigation by light and electron microscopy. Biopsies weremicro-dissected into glomerular (g) and tubulo-interstitial (ti)compartments. The mRNA profiles were obtained using published protocolswith an inter-array reproducibility above 0.98. RNA amplificationensured probe set detection independent of the number of glomeruliisolated. Data stored in the ASBC was processed in a standardizedopen-source analysis pipeline, Genepattern, to generate gene expressionmaps. LPLA2 expression was compared between different glomerulardiseases and analyzed using significance analysis of microarrays (SAM)(Lorz et al., J Am Soc Nephrol. 19(5):904-14, 2008). Significance wasexpressed as a q value which accounts for the false discovery rate ofmultiple samples.

These data show that mRNA expression of LPLA2 is decreased in themicrodissected glomeruli of patients with lupus, compared to theglomeruli of normal controls and glomeruli of patients with glomerulardisease secondary to diabetes, hypertensive nephrosclerosis, minimalchange disease, and IgA nephropathy.

Example 7 Assays for LPLA2 Activity and Autoantibodies in Human Serum

LPLA2 null mice and G2A null mice are indistinguishable with theexception of the development of foam cells in the former knockout model.The inability to locally produce either lyso-phosphatidylcholine or freefatty acid in the lymph node or spleen germinal centers may representthe mechanistic link between these two models of lupus. Alternatively,LPLA2 may represent the first gene product identified as required forthe digestion of apoptotic bodies.

SLE represents a complex clinical syndrome. The lupus syndrome may becomprised of a number of mechanistically distinct but phenotypicallysimilar diseases. LPLA2 represents a newly described lysosomal proteinthat is the long sought acidic, secreted phospholipase A2. The loss ofthis activity in mice results in a very strong phenotype that mimicsmany of the clinical features of lupus. Furthermore, an analysis of theplasma lysosomal protein proteome reveals that LPLA2 circulates in humancirculation. In order to test whether a loss of LPLA2 activity is amarker of active lupus, serum assays were first developed for LPLA2activity and for autoantibodies to LPLA2.

A. Serum Lysosomal Phospholipase A2 Assay

LPLA2 activity is distinct from that of other known phospholipase A2sbased on two primary properties. First, the enzyme displays an acidic pHoptimum of 4.5 and has little detectable activity of pH 7.4. Second, theenzyme acts both as a phospholipase A2 and as a transacylase.N-acetyl-sphingoline (NAS, C2 ceramide) is a preferred lipophilicacceptor for the enzyme. Thus the formation of the fatty acyl ester ofC2 ceramide, 1-O-acyl-N-acetyl-sphingosine, provides a highly specificmeasure of activity and is the principle behind the proposed assay to beused (Shayman, J. A., and Abe, A. Methods Enzymol 311, 105-117, 2000).

To date, LPLA2 assays have been restricted to recombinant enzyme,cultured cells, cell culture supernatants, and tissue homogenates.Several factors may affect the recovery and measurement of LPLA2 inplasma or serum and will need to be explored in the process ofvalidating this assay.

The following assay for the enzyme is designed to determine whetherLPLA2 circulates as a free protein or bound protein, whether cofactorsin plasma or serum modulate LPLA2 activity, whether there are additionalenzymes that metabolize the products of LPLA2 activity found in plasmaor serum, and whether there endogenous lipids in plasma or serum thatwill compete with the assay substrate and thereby inhibit LPLA2activity.

The phospholipids DOPC and phosphatidylethanolamine (PE) along with NASare used in the assay system as donor and acceptor, respectively, of anacyl group. The transacylase activity is determined by analysis of the1-O-acyl-N-acetylsphingosine formation rate. The reaction mixtureconsist of 45 mM sodium citrate (pH 4.5), 10 μg/ml bovine serum albumin,40 μM NAS incorporated into phospholipid liposomes (DOPC/PE /dicetylphosphate/NAS (5:2:1:2 in molar ratio)), and a soluble fraction (0.7-10μg) in a total volume of 500 μl. The reaction is initiated by adding theserum or plasma sample, kept for 5-6 min. at 37° C., and terminated byadding 3 ml of chloroform/methanol (2:1) plus 0.3 ml of 0.9% (w/v) NaCl.The mixture is centrifuged for 5 min at room temperature. The resultantlower layer is transferred into another glass tube and dried down undera stream of nitrogen gas. The dried lipid, dissolved in 40 μl ofchloroform/methanol (2:1) is applied on a high performance thin layerchromatography plate and developed in a solvent system consisting ofchloroform/acetic acid (9:1). The plate was dried down and soaked in 8%(w/v) CuSO₄, 5H₂O, 6.8% (v/v) H₃PO₄, and 32% (v/v) methanol. Theuniformly wet plate is briefly dried down by a hair dryer and charredfor 15 min. in a 150° C. oven. The plate is scanned, and the chemicalmass of the reaction products are estimated by the NIH Image version1.62.

The potential effects of cofactors, protein binding, interfering lipids,or metabolism of product is evaluated by a number of experiments. Theseinclude determining the formation of product over time and as a functionof sample size. In addition, serum samples can be “spiked” with knownamounts of authentic, recombinant LPLA2 to ascertain if interferingsubstances are present. Recombinant LPLA2 can also be assayed in thepresence of albumin or lipoprotein to determine whether these interferewith the assay. The enzyme activity is expressed as amoles 1-O-acyl-NASformed per ml serum per minute. The activities in human serum and plasmaare compared as well as the reproducibility of the assay in the samesample with repeated measurements over time. The effects of storage,heparin, calcium, chelation, and freeze thawing are additional factorsthat are evaluated.

B. Serum Assay for Lysosomal Phospholipase A2 Autoantibodies

The following protocol is employed for the measurement of autoantibodiesto LPLA2. Recombinant LPLA2 (0.1 to 0.5 μg in 50 μl PBS per well) isabsorbed onto microtiter plate wells by overnight incubation of 4° C.The plates are washed 4 times with PBS/0.1% Tween. The plates are thenblocked for 1 hour with 50 μl of PBS with 10 mg/ml bovine serum albumin.Wells are washed with the PBS/Tween solution and then serum samples orcontrols are added for 2 hours at room temperature and then overnight at4° C. The wells are washed again with PBS/Tween and then a secondaryanti-human IgG antibody is added for 2 hours at room temperature.Following the same washing step, detection utilizes O-phenylenediaminedihydrochloride as substrate for horseradish peroxidase. Plate wellabsorbance is read at 492 nm.

Important variables to establish include whether the serum samplesrequire dilution, whether the LPLA2 used for coating the plates shouldbe denatured with carbonate buffer (pH 9.6, 0.05 M), and whether theserum samples should be similarly denatured with carbonate buffer.

The reproducibility of detecting antibodies present, the effects ofserum freezing and storage, and the potential effects of interferingsubstances on the assay are variables that will be considered in thevalidation of this assay.

Example 8 LPLA2 Activity and Autoantibody Titers in Bio-Banked SerumSamples and Correlation of the Clinical Profiles to the SLE DiseaseActivity Index

The Michigan Lupus Cohort (MLC) is a dynamic, prospective cohort of SLEpatients receiving ongoing care at the University of Michigan. The MLCwas established in the year 2000 and currently includes over 600patients (90% female; 79% white, 17% black, 1% Hispanic, 2%Asian/Pacific Islander, 1% other). Cohort participants sign informedconsent which enables systematic follow up of clinical, social anddemographic data. Detailed laboratory and clinical data, includingprospective assessment of disease activity (SLEDAI score) (Bombardier,C. et al. Arthritis Rheum 35, 630-640, 1992), are collected at eachclinic visit, which typically occurs at three month intervals. Arepository of blood samples (which are collected in association withmost clinic visits) has also been prospectively generated since start ofthe MLC. The MLC also serves as a screening and recruiting tool for SLEprotocols, and has facilitated numerous collaborations and ancillaryprojects.

The MLC is the source of patient sera and clinical data for measurementof LPLA2 activity and auto antibody titers. The longitudinal nature andrichness of the clinical data make this an exceptionally valuableresource for this study. Sera is assayed from every patient currentlyparticipating in the MLC. These assays are performed in a blindedfashion ion and samples are randomly assayed without regard for diseaseactivity or clinical phenotype. Age and gender matched samples fromhealthy patients as well as those with the absence of autoimmune disease(e.g. osteoarthritis) are studied as controls. Based on thesemeasurements, a normal range for LPLA2 activity is determined. Forpatients identified that either exhibit significantly lower activitiesof LPLA2 compared to controls or the presence of significant titers ofanti-LPLA2 antibodies, a comprehensive evaluation of their clinicalphenotypes is undertaken. A significant number of MLC patientsexhibiting low LPLA2 activity or elevated anti-LPLA2 demonstrate astatistically significant association between these findings and thepresence of a particular clinical phenotype (e.g. lymphadenopathy) orlaboratory finding (e.g., anti-phospholipid antibodies). For thosepatients that are found to have positive findings, a retrospectivedetermination of LPLA2 activity and antibody titer is made on allsamples available to investigate a correlation with disease activity.The potential association between gender and LPLA2 activity andauto-antibody titers is also evaluated.

Example 9 LPLA2 Activity in an Animal Model

Human or mammalian LPLA2 enzyme, or a variant or derivative thereof istested in an animal model exhibiting an accumulation of tingible bodymacrophages are known, e.g., LPLA2−/− knockout mouse model. Other mousemodels in which such agents are tested for ability to reduceaccumulation of intracellular tingible body macrophages or reduce othersigns and symptoms of autoimmune disease include: the Ro mouse model(Xue et al., Proc Natl Acad Sci USA 100: 7503-7508, 2003), the Tyro 3mouse model (Lu et al., Science 293: 306-311, 2001), the c-mer mousemodel (Cohen et al., J Exp Med 196: 135-140, 2002), and the MFG-E8 mousemodel (Hanayama et al., Science 304: 1147-1150, 2004).

The references cited herein throughout, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are all specifically incorporated herein by reference.

1. A method of reducing accumulation of intracellular tingible bodymacrophages comprising the step of contacting a cell having anaccumulation of tingible body macrophages with an agent having lysosomalphospholipase A2 (LPLA2) enzymatic activity in an amount effective toreduce the accumulation of tingible body macrophages.
 2. The method ofclaim 1 wherein the agent having LPLA2 enzymatic activity is a proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-288.
 3. The method of claim 1 wherein said agent is amammalian LPLA2 enzyme.
 4. The method of claim 1 wherein said agent is ahuman LPLA2 enzyme.
 5. The method of claim 1 wherein said agent is anLPLA2 enzyme comprising one or more mannose residues.
 6. The method ofclaim 4 wherein said human LPLA2 enzyme comprises the amino acidsequence set out in SEQ ID NO: 1, or a fragment thereof, derivative orvariant of SEQ ID NO: 1 having LPLA2 enzymatic activity.
 7. The methodof claim 6 wherein said human LPLA2 enzyme comprises the catalytic sitecorresponding to amino acids 196-200 (A-X-S-X-G) of SEQ ID NO:
 1. 8. Themethod of claim 7 wherein said human LPLA2 enzyme further comprises acysteine bond corresponding to the cysteine bond between the Cys atposition 65 and the Cys 89 of SEQ ID NO:
 1. 9. The method of claim 7wherein said human LPLA2 enzyme comprises an amino acid sequence havingat least 80% identity to the amino acid sequence of a fragment of SEQ IDNO: 1 that is at least 75 residues in length and that comprises aminoacids 196-200 of SEQ ID NO:
 1. 10. The method of claim 3 wherein saidmammalian LPLA2 enzyme is a protein comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-288 and a fragment,derivative and variant of SEQ ID NOs: 1-288.
 11. The method of claim 10wherein said LPLA2 enzyme comprises the catalytic consensus sequence-A-X-S-X-G-.
 12. A method of treating a patient diagnosed with adisorder characterized by the intracellular accumulation of tingiblebody macrophages comprising administering to said patient an effectiveamount of an agent having LPLA2 enzymatic activity.
 13. The method ofclaim 12 wherein the disorder is systemic lupus erythematosus.
 14. Themethod of claim 12 wherein the disorder is drug-induced lupus.
 15. Themethod of any of claims 12-14 wherein the agent having LPLA2 enzymaticactivity is a protein comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-288.
 16. The method of any ofclaims 12-14 wherein said agent is a mammalian LPLA2 enzyme.
 17. Themethod of any of claims 12-14 wherein said agent is a human LPLA2enzyme.
 18. The method of claim any of claims 12-14 wherein said agentis an LPLA2 enzyme comprising one or more mannose residues.
 19. Themethod of claim 18 wherein said human LPLA2 enzyme comprises the aminoacid sequence set out in SEQ ID NO: 1, or a fragment thereof, derivativeor variant of SEQ ID NO: 1 having LPLA2 enzymatic activity.
 20. Themethod of claim 19 wherein said human LPLA2 enzyme comprises thecatalytic site corresponding to amino acids 196-200 (A-X-S-X-G) of SEQID NO:
 1. 21. The method of claim 20 wherein said human LPLA2 enzymefurther comprises a cysteine bond corresponding to the cysteine bondbetween the Cys at position 65 and the Cys 89 of SEQ ID NO:
 1. 22. Themethod of claim 20 wherein said human LPLA2 enzyme comprises an aminoacid sequence having at least 80% identity to the amino acid sequence ofa fragment of SEQ ID NO: 1 that is at least 75 residues in length andthat comprises amino acids 196-200 of SEQ ID NO:
 1. 23. The method ofclaim 16 wherein said mammalian LPLA2 enzyme is a protein comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:1-288 and a fragment, derivative and variant of SEQ ID NOs: 1-288. 24.The method of claim 23 wherein said LPLA2 enzyme comprises the catalyticconsensus sequence -A-X-S-X-G-.
 25. A method of screening for apotential therapeutic that decreases intracellular levels of tingiblebody macrophages comprising the step of measuring the number orphagocytic index of intracellular tingible body macrophages in a lupusmouse model in the presence and absence of a test compound, wherein adecreased intracellular tingible body macrophage level in the presenceof the test compound compared to an intracellular tangible bodymacrophage level in the absence of the test compound identifies the testcompound as a potential therapeutic, and wherein the test compound is afragment, variant or derivative of SEQ ID NO: 1-288.
 26. A method ofscreening for a potential therapeutic that decreases intracellularlevels of tingible body macrophages comprising the step of measuringLPLA2 enzyme activity in the presence and absence of a test compound,wherein an increased level of LPLA2 enzyme activity in the presence ofthe test compound compared to the absence of the test compoundidentifies the test compound as a potential therapeutic.
 27. A methodfor determining susceptibility to systemic lupus erythematosus in anindividual comprising the step of determining lysosomal phospholipase A2(LPLA2) enzymatic activity in a sample from an individual, or the stepof determining LPLA2 expression in a sample from an individual, whereina decreased LPLA2 enzymatic activity or LPLA2 expression level in theindividual compared to LPLA2 enzymatic activity or LPLA2 expressionlevel in a normal individual indicates susceptibility to systemic lupuserythematosus.
 28. The method of claim 27 wherein a decreased LPLA2enzymatic activity or LPLA2 expression level compared to prior LPLA2enzymatic activity or LPLA2 expression level in the same individualindicates susceptibility to systemic lupus erythematosus.
 29. A methodfor diagnosing systemic lupus erythematosus comprising the step ofdetecting lysosomal phospholipase A2 (LPLA2) autoantibodies in a samplefrom an individual, wherein detecting increased levels of LPLA2autoantibodies in the individual compared to the level of LPLA2autoantibodies in a normal individual is suggestive of systemic lupuserythematosus diagnosis, wherein said normal individual is known not tosuffer from systemic lupus erythematosus.
 30. The method of claim 29wherein detecting an increased level of LPLA2 autoantibodies in a samplefrom an individual compared to a prior level of LPLA2 autoantibodies inthe same individual is suggestive of a diagnosis of systemic lupuserythematosus.
 31. A method for determining susceptibility to systemiclupus erythematosus in an individual comprising the step of detectinglysosomal phospholipase A2 (LPLA2) autoantibodies in a sample from anindividual, wherein a detecting increased levels of LPLA2 autoantibodiesin the individual compared to the level of LPLA2 autoantibodies in anormal individual is suggestive of systemic lupus erythematosussusceptibility, wherein said normal individual is known not to sufferfrom systemic lupus erythematosus.
 32. The method of claim 31 whereindetecting an increased level of LPLA2 autoantibodies in a sample from anindividual compared to a prior level of LPLA2 autoantibodies in the sameindividual is suggestive of susceptibility to systemic lupuserythematosus.
 33. A method for determining the progression of systemiclupus erythematosus in an individual comprising the step of determininglysosomal phospholipase A2 (LPLA2) enzymatic activity in samples fromthe individual taken over time wherein a decrease in LPLA2 enzymaticactivity in the samples taken over time in the individual is suggestiveof progression of systemic lupus erythematosus.
 34. A method fordetermining the progression of systemic lupus erythematosus in anindividual comprising the step of detecting lysosomal phospholipase A2(LPLA2) autoantibodies in samples from the individual taken over timewherein an increase in LPLA2 autoantibodies in the samples taken overtime in the individual is suggestive of progression of systemic lupuserythematosus.
 35. The method of claim 1 or claim 12 further comprisingtreating patients having a polymorphism that reduces expression oractivity of LPLA2.
 36. The method of claim 35 wherein the polymorphismis at a position corresponding to amino acids 196-200 of SEQ ID NO: 1.